(navigation image)
Home American Libraries | Canadian Libraries | Universal Library | Community Texts | Project Gutenberg | Children's Library | Biodiversity Heritage Library | Additional Collections
Search: Advanced Search
Anonymous User (login or join us)
Upload
See other formats

Full text of "Review of intramural research 1961"

fl863 



NATIONAL INSTITUTES OF HEALTH 



Review 
of 



INTRAMURAL 

RESEARCH 

1961 



LIBRARY 

NOV 41963 

MATiOHAL \mmiii OF HEALTH 




U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 

Public Health Service 



ISSUED IN A LIMITED EDITION FOR ADMINISTRATIVE USE 



NATIONAL INSTITUTES OF HEALTH 



Review 
of 

INTRAMURAL 
RESEARCH 

1961 




U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 

Public Health Service 
National Institutes of Health, Bethesda 14, Maryland 



INSTITUTE RESEARCH DIRECTORS 

National Cancer Institute Charles G. Zubrod, M.D. 

Scientific Director 

Nathaniel I. Berlin, AI.D. 

Clinical Director 

National Heart Institute Robert W. Berliner, M.D. 

Scientific Director 

Donald S. Fredrickson, M.D. 

Clinical Director 
National Institute of Arthritis 

AND Metabolic Diseases De Witt Stetten, M.D. 

Scientific Director 

Joseph J. Bunim, M.D. 

Clinical Director 

National Institute of Allergy 

AND Infectious Diseases Dorland J. Davis, M.D. 

Scientific Director 

Vernon Knight, M.D. 

Clinical Director 

National Institute of Mental Health John C. Eberhart, M.D. 

Scientific Director 

Eobert A. Cohen, M.D. 

Clinical Director 
National Institute of Neurological 

Diseases and Blindness G. Milton Shy, M.D. 

Scientific Director 

Maitland Baldwin, M.D. 

Clinical Director 

National Institute of Dental Research .... Seymour Kreshover, M.D., D.D.S. 

Scientific Director 

Dr. Edward J. Driscoll, D.D.S. 

Division of Biologics Standards Roderick Murray, M.D. 

Scientific Director 



G. Burroughs Mider, M.D. 
Director of Laboratories and Clinics 

Thomas J. Kennedy, M.D. 
Assistant to Director of Laboratories and Clinics 



m 



FOREWORD 

The National Institutes of Health, one of the five Bureaus of the United States Public 
Health Service, has been assigned the mission to conduct and to support research, re- 
search trainmg, and other related activities. Tliis mission is discharged partially- 
through an extramural suppox-t program wliich, administered fi'om Bethesda, reaches 
into virtually every institution ui the United States engaged in biomedical research, and 
is rapidly expanding throughout the world. And it is partially discharged through a 
direct operation, the intramural program, housed in laboratories in Bethesda, and repre- 
senting somethuig of a microcosm of the total effort. This publication focuses exclu- 
sively on the intramural enterprise, describing it in a series of revie'ws designed to 
illustrate the compass and flavor of the local research activities of each Institute. Thus, 
it is not a comprehensive presentation of the program of either the United States Public 
Health Service or of the National Institutes of Health. 

Glimpses of the extent to which the uitramural research effort has participated in 
the broad forward thrust of medical research are provided in the eight Annual Sum- 
mary Reports comprising this compendiimi. The reports are protected from editorial 
interference, and prepai-ed by the originators in accord with onJy the most general guide- 
lines. This procedure, considered and deliberate, has been adopted to aEow the reader 
to savor the diversity of outlook and attitude which prevails among a gi'oup of brilliant 
scientists loosely knit and almost imperceptibly harnessed for the attainment of common 
categorical goals. In such a presentation, it is possible to overlook the thread of mission 
that ties each of these operating research organizations into the bi'oad program of the 
National Institutes of Health. It is appropriate, therefore, to pursue this aspect briefly. 

The mission of the National Institutes of Health as a whole and of its components 
is to develop the facilities, resources, and attitudes most effective in acquiring new 
knowledge concerning disease processes, and relieving suffering, bringing about cure 
and rehabilitation, and assuring the prevention, whenever possible, of disease. Broadly 
considered, this mission involves providing the wherewithal and cultivating suitable soil 
for a systematic study of man and Ms milieu with the ultimate objective of contributing 
to improved health. From this overall point of view, the NIH does not differentiate 
between what is done intramurally and extramurally. However, witliin these concep- 
tions are contained more specific objectives only some of wliich can be sought for within 
an intramural research program, while the others may be searched out most expedi- 
tiously by support of work in other uistitutions through the extramural program. 

This second Annual Review of NIH Intramural Research provides evidence of the 
magnitude of the effort — ^both in breadth and depth — and of the type of acliievements 
that have placed this installation in the forefront of research in the medical sciences. 



CONTENTS 



Page 

forewokd v 

National Cancer Institute 1 

Introduction 1 

The Leukemias 1 

Causation 1 

Pathogenesis and bodily economy 2 

Management of the leukemias 3 

Biochemical and physiological studies of clinical 

cancer 5 

Nucleic acids 5 

Amino acids 6 

Plasma proteins 6 

Biochemistry of the skin 7 

Endocrine studies 7 

Hematopoiesis ^ 8 

Folic acid antagonists 9 

Metabohc balance technique 9 

Calcium metaboHsm 10 

Serum lactic dehydrogenase 10 

Motosis-inhibiting activity in the serum ... 10 

Carcinoid 10 

Immunology 10 

Metabolism service 11 

Amino acid transport 11 

Plasma proteins 11 

Proteins in normal and malignant plasma 

ceUs 11 

Metabolic behavior 12 

Immunologic studies 13 

Nucleic acids 13 

Calcium metabolism 13 

Porphyrin metabolism 14 

Erythropoiesis 14 

Erythropoietin production . 14 

Red-cell hfe span methodology 15 

Clinical studies of erythropoiesis .... 15 

Laboratory of biochemistry 15 

Cytochemistry section 15 

Nucleic acids section 16 

Nutrition and carcinogenesis section .... IS 

Protein chemistry section 19 

Tumor-host relations 21 

Laboratory of biology 23 

Carcinogenesis 23 

Immunology 24 

Virology 25 

Genetics 26 

Drug resistance 27 



Pago 

Laboratory of chemical pharmacologj' 27 

Immunology section 28 

The tumor-antigen problem 28 

Role of the antigen in immunologic 

tolleranee 28 

Endotoxin 28 

Natural antibodies 30 

Macromoleeular chemistry section 32 

Synthesis and structure 32 

Enzyme inhibition with polyeleetrolytes . 33 
Inactivation of S. marcescens polysac- 
charide 34 

Bacteriophage 34 

Virus inactivation with polyeleetro- 
lytes 35 

Biochemical pharmacology section 35 

Immunity 35 

Virus leukemia 37 

CNS tumors 37 

Evaluation of antitumor agents .... 38 

FoHc acid antagonists: mechanisms ... 39 

Drug resistance 40 

Other topics 42 

Dermatology branch 42 

Epidermal growth and differentiation .... 42 

Mycosis fungoides 43 

Endocrinology branch 43 

Medicine branch 45 

Chemotherapy service 45 

Clinical pharmacology and experimental thera- 
peutics service 46 

Laboratory of pathology 47 

Collaborative research 47 

Laboratory animals 48 

Transplantable tumors 48 

Virus research 49 

Chemical carcinogenesis 50 

Geographic pathology 50 

Plasma-cell neoplasms in mice 50 

Pathologic anatomy branch 51 

Laboratory of physiology 51 

Radiation branch 54 

Surgery branch 55 

Local wound chemotherapy 55 

Tumor growth and metastases 57 

Virus and surgical treatment of cervical 

cancer 57 

Head and neck cancer 58 

Cancer of uterine cervix 58 



vin 



CONTENTS 



Page 

Laboratory of viral oncology 58 

Viruses in relation to cancer 59 

Laboratory animal systems 59 

Murine leukemia viruses 59 

Polyoma virus 61 

Rous sarcoma virus 62 

Other viruses important to tumor- 
virus research 63 

Indigenous viruses 63 

Viruses in human neoplasias 64 

Human cancerous tissues 64 

Specimens from surgical opera- 
tions 64 

Specimens derived at autopsy. . 65 
Human leukemic blood and bone 

marrow 65 

Other human lesions 66 

Tissue culture 66 

General 66 

In vitro cell biology studies 66 

Nutritional and metabohc 66 

Malignant transformation in vitro . . 67 

Instrumentation and methods 67 

Other areas of investigation 68 

Carcinogenesis 68 

DiflFusion-chamber induction of 

plasma-ceU turmors 68 

Ultrastructure of plasma-cell tumors . 68 
Transmission of hamster tumors by 

feeding of tumor cells 69 

Immunology and serology 69 

Radiation 70 

National Heabt Institute 71 

Introduction 71 

Laboratory of cellular physiology and metabolism . 71 

Section on cellular physiology 71 

Section on metabolism 74 

Pathway and inhibitors of cholesterol 

biosynthesis 74 

MetaboHsm of cholesterol esters .... 74 
Effects of dietary fat on cholesterol metab- 

ohsm 75 

Lipidoses and hyperlipemia 75 

Metabolism of adipose tissue and hormo- 
nal effects on fat mobilization .... 76 
Metabohc fate of free fatty acids .... 77 

Phosphohpid metaboUsm 78 

Metabolism of rioimoleic acid and other 

hydroxy fatty acids 78 

Protein metablism 78 

Section on enzymes 79 

One carbon metaboUsm 79 

Formate activation 79 

Carbon dioxide activation 80 

Carboxylation of aoetyl-CoA .... 80 

Two-carbon metabolism 81 

Fatty-acid synthesis 81 

Ethylene glycol metaboUsm .... 81 

Acetyl cyanide formation 82 



Fag* 

Laboratory of cellular physiology-etc. — Continued 
Section on cellular physiology — Continued 

MetaboUsm of heterocycUc compounds . 82 

Riboflavin degradation 82 

Nicotinic acid fermentation .... 82 
Phenazine-1-carboxyUc acid biosyn- 
thesis 83 

MetaboUsm of amino acids 83 

Glycine reduction 83 

Lysine degradation 83 

Gamma-aminobutyrate fermentation . 83 

MetaboUsm of onium compounds .... 83 

Choline fermentation 83 

Fermentation of sulfonium com- 
pounds 84 

Transsulfuration 84 

Laboratory of chemistry of natural products ... 85 

Gas phase chromatographic methodology . . 85 

Alkaloid work 85 

The KaUikrein-Kallidinogen-KalUdin system . 86 

Laboratory of chemical pharmacology 86 

Interaction of drugs with physiological and 

biochemical function 86 

Amine storage site 86 

Control of function by drugs acting on 

storage sites 87 

Role of 5 HT and NE in brain 88 

Amines as modulating agents 88 

Biochemical behavior 89 

Central control of energy substrates ... 89 

Central control of metaboUsm 89 

Desmethylimipramine, a new antidepressant . 90 

Passage of substances across membraines . . 90 

Ca in membrane permeability 90 

Penetration of drugs into ceUs 90 

Biliary excretion of drugs 90 

Membranes within the CNS 90 

Transport of purines and pyrimidines . . 91 

Drug metaboUsm 91 

Antimetabolites metabolized by enzymes 

of intermediay metaboUsm 91 

Substances acted on by non-specific en- 
zymes 91 

Metabolism of imipramine 92 

Deposition of calcium 92 

Deposition of triglycerides 92 

Development of new drugs 92 

Development of new methods of analysis . . 93 

Laboratory of technical development 93 

Gas chromatography 93 

Determination of blood gases 94 

Radioassay: scintillation counting ... 94 

Radioassay: ionization chamber .... 95 

Analysis of free fatty acids 95 

Porphyrin 96 

Blood-flow measurement 96 

Ultrasonics 96 

Theoretical analysis of transport 97 

Fluorescence and phosphorescence 97 

Freezing-point determinations 97 



CONTENTS 



DC 



Page 

Laboratory of cardiovascular physiology 98 

Electrolyte heart changes during homeometric 

autoregulation gg 

Isolated papillary muscle gg 

Afferent pathways influencing efferent auto- 
nomic discharge gg 

Carotid body hypoxia 9g 

Central nervous system hypoxia .... 99 
Effect of carotid hypotension on renal 

blood flow 99 

Vagal afferent pathways 99 

Dynamics of the cardiac cycle 100 

The ventricle 100 

The atrium 100 

Electrophysiological studies 100 

Mj'ocardial catechol amines 101 

Vasculature during exercise 102 

The Ivallikrein system 102 

Laboratory of kidney and electrolyte metabolism . 102 

Renal physiology 102 

Micropunctiire studies 102 

Carbonic anhydrase in rat renal cortex . 103 

Measurement of medullary blood flow . . 103 

Diuretic agents 104 

Phosphate secretion 104 

Electrolyte and water transport 104 

Renal cortical slices and tubule suspen- 
sions 104 

Sodium andpotassium dependent ATPase . 105 

Cation transport 105 

Alterations in lipid content .... 105 

Transport in rat red cells 105 

Divalent cations 105 

Water movement across the toad bladder . 106 

Vasopressin 106 

Estimation of vasopressin in biological 

fluids 107 

Effect of solute concentration on net water 

movement . 107 

Water movement across an artificial liquid 

membrane 107 

Aldosterone 108 

Aldosterone stimulating hormone ... 108 

Renin-angiotensin system 108 

Formation of ASH 109 

Sensitization to aldosterons 109 

Disappearance from plasma 109 

Cardiac glycosides 109 

Cardioglobulin and related studies 109 

Mammalian hearts 109 

Degradation of cardioglobuUn 110 

Ryanodine-digitalis antagonism .... 110 

Laboratory of cHnical biochenaistry 110 

Amine biogenesis and metabolism 110 

Homocarnosine and carnosine metabolism . . 113 

Choline biogenesis 113 

Collagen and hydroxyproline 113 

Amino-acid transport 114 

Biosynthesis of the B12 coenzyme 114 

643351—62 2 



Fagt 

Clinical endocrinology branch . . . , 114 

Experimental therapeutics branch 117 

Biosynthesis and metabolism of aromatic 

amines ng 

Action and metaboUsm of drugs 118 

Metabolism of hydroxyproline and coUagen . 119 

Miscellaneous 120 

Cardiology branch 120 

Dynamics of ventricular contraction .... 120 
Adrenergic nervous system and myocardial 

function 121 

Cardiovascular diagnostic techniques .... 123 

CUnical cardiology 125 

Circulatory physiology 126 

Clinical physiology 127 

CUnical biophysics 127 

Instrumentation 127 

Circulatory studies 128 

Pulmonary studies 128 

Surgery branch 128 

Gerontology branch 133 

National Institute op Allergy and Infectious 

Diseases 141 

Introduction 141 

Laboratory of clinical investigation 143 

Viral infection of volunteers 143 

Salmonella carriers 144 

Malaria in volunteers 144 

Mechanisms of fever 144 

Fungus disease 144 

Penicillin derivatives 145 

Penicillin-resistant staphj'lococoal infections . 145 

Clinical immunology 146 

Mouse-plasma cell tumors 146 

Laboratory of immunology 146 

Passive transfer of allergic encephalomyelitis . 146 
Antibody production and gamma globulin in 

human malaria 146 

Proteolytic enzyme of human spleen purified . 146 
Lupus serum reacts with mammalian chromo- 
somes 147 

Genetic control of gamma globulin allotypes . 147 

Allergic thyroiditis 147 

Laboratory of bacterial diseases 147 

In vitro cell antibody 147 

Staphylococcus virulence 148 

Brucellosis diagnosis 148 

Laboratory of biology of viruses 148 

Virus-host relationship 148 

Virus structure 149 

Virus inhibitors 149 

"Foreign" ceU antigen 149 

Melanin granules and mitochondria 149 

Laboratory of parasite chemotherapy 149 

Malaria-simian 150 

Malaria studies in Malaya 150 

Malaria-human 150 

Biochemical studies 150 



CONTENTS 



Page 
Laboratory of parasite cliemotherapy— Continued 

Nutrition and malaria 1^" 

Intestinal parasites ^^^ 

Insect tissue culture l^l 

Virus-mosquito larvae associations 151 

Virus-parasite combinations 151 

Laboratory of parasitic diseases 151 

Entamoeba histolytica 151 

Freezing protozoa l^'^ 

Nematode cycle in germ-free animals .... 152 



Nutrition and schistosomiasis 



152 



Biochemical studies 153 

Virus potentiation by trichinella 153 

Toxoplasmosis ^^^ 

Laboratory of tropical virology 154 

Equine encephalitis 154 

Vesicular stomatitis virus 154 

Virus from rain forest arthropods 155 



Mycoses . 



155 



156 
156 
157 
157 
158 



Rocky mountain laboratory 155 

Tularemia ^^5 

Disease of wildlife 156 

Arthropod-borne viruses 

Q fever 

Rocky mountain spotted fever 

Allergic phenomena 

Endotoxins 

Microorganisms 15° 

Radioisotope precipitation 158 

Laboratory of germ-free animal research 159 

Susceptibility by sex 159 

Helminth infections 159 

"Natural" antibodies 159 

Mouse colony free of virus 160 

Thyroiditis in guinea pig 160 

Trauma in amoebic infections 160 

Second generation guinea pigs 161 

Germ-free chickens 161 

Laboratory of infectious diseases 161 

Respiratory virus disease 161 

Eaton agent is PPLO 161 

Enteroviruses in acute respiratory disease . . 162 

Antibiotics in acute respiratory disease ... 162 

Respiratory viruses in human volunteers . . 162 

Vaccine development 163 

Human viral diseases 163 

Cancer viruses 163 

Natural history of polyoma vh-us 163 

Cancer-virus study 164 

SPF and germ-free mice 164 

Fungus disease 164 

Penicillinase 164 

Antibacterial marine waters 165 

Staphylococcus iron metaboUsm 165 

Cell division in streptococci 165 

Antibacterial substances in mollusks .... 166 

Hydrogenomonas 166 

Eosinophilic meningitis 166 



National Institute of Arthritis and Metabolic 
Diseases 

Basic Research 

Introduction 

Genetics 

Enzyme-catalyzed reactions 

General 

Laboratory of experimental pathology 

Cell structures in aging 

Cytogenetic studies 

Degenerative joint disease 

Hematology 

Hemoglobin 

Histochemistry 

Human pathology 

Hypersensitivity 

Immunochemistry 

Marrow cultures 

Melanomas 

Nutritional deficiencies 

Serum enzyme changes after stress 

Pneumocystis carinii infection 

Laboratory of biochemistry and metaboHsm . . . 

Carbohydrate metabolism 

Polymers 

Small molecules and coenzymes .... 

Biosynthesis 

Thiamine 

Lipid synthesis 

Regulatory mechanisms and hormones . . . 

Insulin 

Humoral agents in uremia 

Pyrophosphatase 

Galactosemia 

Steroid hormones 

Nucleic acids 

Cell-free protein synthesis 

Lysogeny 

Histidine biosynthesis 

Model compounds 

Laboratory of chemistry 

Carbohydrates 

Medicinal chemistry 

Metabolites 

Steroids 

Microanalytical services 

Laboratory of molecular biology 

Laboratory of nutrition and endocrinology . . . 

Folic acid 

Vitamin B12 

Dietary liver necrosis 

Factor 3-selenium compounds 

Vitamin E antioxidants and selenium . . . 

Other fat soluble vitamin studies 

Plasmalogens 

Protein 

Experimental obesity 

Germ-free animal research 



I'age 



167 

167 

167 

167 

168 

16S 

168 

168 

168 

169 

169 

169 

170 

171 

171 

171 

171 

171 

171 

172 

172 

172 

172 

173 

173 

173 

173 

174 

174 

174 

174 

174 

174 

175 

176 

176 

176 

176 

177 

177 

178 

179 

183 

184 

184 

185 

185 

186 

186 

186 

187 

187 

187 

187 

188 

188 



CONTENTS 



Page 
Laboratory of nutrition and endocrinology — Con. 

Guinea pig nutrition 189 

Glucose tolerance factor 189 

High calcium intake 189 

Riboflavin 189 

Adipose tissue metabolism 190 

Protein hormones 190 

Laboratory of pharmacology and toxicology .... 191 

Spermidine and spermine 191 

Sialic acids 191 

Sulfur amino acids 192 

Enzyme levels 192 

Burns 192 

Imidazoles 192 

Leprosy 193 

Mevalonic acid 193 

Enzyme activitj' 193 

Laboratory of physical biology 193 

Office of mathematical research 197 

Clinical Investigations 198 

Arthritis and rheumatism branch 199 

Demography of rheumatic diseases 199 

National examination arthritis survey . . 199 

Indian survey 200 

Sjogren's syndrome 201 

Program planning 203 

Glutamic dehydrogenase 203 

Physical state of enzymes 203 

Amino acids 203 

Dissociation by steroids 204 

Aromatic amino acids and homogentisic acid . 205 

Phenylketonuria 205 

Tyrosine and ascorbic acid 205 

Connective tissue and ochronosis .... 206 

Alcaptonuria 206 

Gout 207 

Uric acid 207 

Microcrystalline urates 207 

Cystinosis 208 

Anti-rheumatic drugs 208 

Gastro-enterology unit 209 

Whipple's disease 209 

Hypogamma globulinemia 210 

Hypolipidemia 210 

Inborn errors of metabolism 210 

Bniverdin reductase 211 

Clinical endocrinology branch 211 

Carbohydrate metabolism 211 

Glucose 211 

Hypoglycemia 212 

InsuUn 212 

Glycogen storage disease 213 

Galactose and galactosemia 213 

Amino acid Transport and protein synthesis . 213 

Biochemistry of the thyroid 214 

Iodide Transport 214 

lodoproteins 214 

Thyroxine and iodotyrosine synthesis . . 214 



XI 
Page 



Clinical endocrinology branch — Continued 
Biochemistry of the thyroid — Continued 

Dosimetry of I'" radiation 216 

Physical chemistry of proteins 216 

Metabolic diseases branch 216 

Mineral metaboUsm 216 

Osteoporosis 216 

Bone metabolism 217 

Energy metabolism 218 

Obesity 218 

Cold and body fat content 220 

Periodic fever 220 

Hematology unit 220 

Blood diseases 220 

Neonatal thrombocytopenic purpura . . 220 

Platelet antigen systems 220 

Platelet transfusions 221 

Antibodies on cell surfaces 221 

Anti-hemophllic factors 221 

Gamma-globulin 222 

"Refractory" anemias 222 

Pediatric metabolism branch 222 

Cystic fibrosis of the pancreas 223 

Genetics and electrolyte abnormality .... 223 

Mucus and mucopolysaccharide 224 

Pulmonary involvement and intestinal malab- 
sorption 224 

Glycogen storage disease 224 

Intestinal malabsorption 225 

National Institute of Dental Research . . 227 

Introduction 227 

Major program areas 228 

Human genetics 228 

Periodontal disease 228 

Nutritional aspects of oral disease .... 229 

Bacteriological aspects of oral disease , . 230 

Electron and X-ray microscopy 231 

Program areas with future promise 232 

Minor program areas 232 

Laboratory of microbiology 233 

Laboratory of biochemistry 335 

Enzyme chemistry 235 

Experimental dental caries 235 

Saliva chemistry 236 

Protein chemistry 236 

Calcification and strontium studies 237 

Laboratory of histology and pathology 237 

Epidemiology and biometry branch 240 

Nutrition surveys 240 

Fluorine and dental caries 341 

Other studies 242 

Clinical investigations branch 243 

Introduction 243 

Oral diseases 243 

Oral pharyngeal development and function . . 245 

Medical investigations 247 

Human genetics 247 



xn 



CONTENTS 



Page 
National Institute of Neurological Diseases 

AND Blindness 251 

Introduction: a decade of research progress . . . 251 

Action potential transmission 251 

Molecular studies and synthesis 252 

Enzymes and intermediate metabolism .... 252 

Ultrastructure 253 

Basic studies in audition 253 

Anatomical studies 254 

Pathology of the nervous system 254 

Physiology of cell aggregations 254 

Embryology 255 

Regeneration 255 

Pharmacological studies 255 

Applied research 256 

New diseases and new concepts 258 

Instrumentation 259 

Summary 259 

Laboratory of neuroanatomical sciences 259 

Introduction 259 

Eighth cranial nerve 260 

Ultrastructure studies in the nervous system . 261 

Regeneration and reinnervation 261 

Embyronic manipulation 262 

Experimental and comparative neuropath- 
ology 263 

Laboratory of biophysics 263 

Laboratory of neurochemistry 266 

Laboratory of neuropathology 269 

Laboratory of neurophysiology 270 

Branch of electroencephalography and clinical 

neurophysiology 271 

Routine diagnostic service 271 

Research activity 272 

Other activities 273 

Medical neurology branch 273 

Introduction 273 

Neuromuscular disorders — anatomy and physi- 
ology 273 

Neuromuscular disorders — pharmacology. . . 275 

Cystic fibrosis of the pancreas 276 

Genetics 276 

Brain tumor studies 276 

Ophthalmology branch 277 

Surgical neurology branch 284 

Introduction 284 

Epilepsy 285 

Involuntary movements 286 

Prenatal lesions 286 

Effects of surgical lesions 286 

Cerebral edema 287 

Deep hypothermia 287 

Effects of radiant energy 288 

Technical development 288 

National Institutb op Mental Health .... 291 

Introduction 291 

Clinical investigations 292 



Page 

Laboratory of clinical science 297 

Schizophrenia 297 

Biochemistry 297 

Psychopharmacology 298 

Psychophysiology 298 

Family studies 298 

The biogenic amines in mental state .... 298 

Experimental allergic encephalomyeUtis . . . 299 

Encephalitogenic protein 299 

Immunologic studies 299 

Thyroxine 299 

Geriatric psychiatry 300 

Catecholamines 300 

Studies in animals 300 

Studies in man 301 

Sleep and arousal 301 

Laboratory of psychology 302 

Early development 302 

Perception 305 

Social communication 305 

Perceptual functions of posterior association 

cortex 308 

Problem-solving functions of frontal associa- 
tion cortex 309 

Reticular activating system 309 

Limbic system 310 

Aging 311 

Schizophrenia 213 

Stress 314 

Laboratory of socio-environmental studies .... 315 

The family 316 

Cross-national comparisons 316 

Social class and parent-child relationships . 316 

Effects of maternal employment .... 316 

The childs' development of conscience . . 317 

Methodological investigations 317 

The mental hospital 318 

Cross-national comparisons 318 

The mental hospital in the United States . 318 

The social behavior of patients ..... 320 

Relevant social variables 320 

Self-esteem among adolescents 320 

Social psychologj'' of aging 321 

Genetics and environment 322 

Addiction research center 322 

General 322 

Addictive properties of new analgesics .... 324 

Four new synthetic compounds 324 

Acute and chronic intoxication 325 

Clinical studies of intoxication 326 

Biochemistry of addiction 326 

Chronic intoxication with barbiturates . . . 327 

Psychological studies of addiction 329 

Central nervous system depressants 331 

Conditioning factors in addiction and habitua- 
tion 332 

Effects of drugs on "mental set" 334 

Laboratory of cellular pharmacology 335 



CONTENTS 



xni 



Page 

Laboratory of neurobiology 338 

Scientific program 338 

Physical analysis of excitability .... 338 

Neurophysiology of gUa and dendrites . . 338 

Brightness discrimination 338 

Prolonged sensory stimulation 339 

Sensory and cerebello-cerebral path- 
ways 339 

Sensorimeter conduction systems .... 339 

Academic activities 340 

Laboratory of neurochemistry 340 

Section on physical chemistry 340 

Laboratory of neurophysiology 342 

Adult psychiatry branch 344 

Child research branch 356 

Program summary 356 

Behavior continuities in the infant and pre- 
school child 357 

Interpersonal patterns of adaptation .... 359 

Methods 359 

Marital relationships 359 

Pregnancy study 360 

Matrix of variables 361 

Comment 361 



Page 

Cluneal neuropharmacology research center . . . 362 

Development of an admission service .... 362 

Social contact in a psychiatric ward 363 

Schizophrenia 364 

Psychoactive tryptamine derivatives .... 365 

Chlorpromazine 366 

Epinephrine metabohtes 366 

Brain stem neurones 367 

Animal behavior studies 368 

Division or Biologics Standards 371 

Introduction 371 

Laboratory of control activities 372 

Laboratory of bacterial products 373 

Laboratory of viral immunology 374 

Laboratory of virology and rickettsiology .... 376 

Simian virus 40 376 

Measles 377 

Homologous serum jaundice 377 

Typhus 378 

New antimicrobial agents 378 

Intracellular infections 378 

Laboratory of biophysics and biochemistry . . . 379 

Laboratory of blood and blood products 379 

Cooperative research 380 



NATIONAL CANCER INSTITUTE 



INTRODUCTION 

In. this report I have commented on a particular 
area in order to illustrate some of the directions 
and problems of current research upon cancer at 
the National Cancer Institute. The very simplic- 
ity of the name of the Institute's categorical mis- 
sion, "Cancer," disguises the biggest problem — 
that like the term "Infection," it includes a great 
diversity of biological, biochemical, and disease 
phenomena. In recent years it has become ever 
more apparent that generalizations about cancer 
in man and animals are unwise and that research 
at this time must focus upon fragments. The 
success enjoyed by Institute scientists in chorio- 
carcinoma emphasizes the specificity of cancer 
research. 

As one reads through the amiual reports, one 
becomes aware of the extent of interest in the 
leukemias. In almost every laboratory and branch 
some aspect of the leukemias is being studied, and 
it is fair to say that research in these diseases con- 
stitutes a major NCI commitment. The following 
remarks will survey some of the Institute's pro- 
grams on the causation, development, and manage- 
ment of the leukemias. 



THE LEUKEMIAS 

Causation 

In animals and man tlie leukemias arise "spon- 
taneously," that is, by some unknown stimulus, or 
follow exposure to several types of irradiation. 
In mice it has been shown that most types of spon- 
taneous leukemias are due to viruses. New re- 
search findings have increased understanding of 
some of these phenomena during the past year. 

One of the major barriers to the demonstration 
of the viral causation of spontaneous animal 
tumors has been the relatively low concentration 
of virus in the tumor-bearing animal. A whole 
series of developments, to which NCI scientists 
have made major contributions, now permits the 



isolation and identification of these small amounts 
of virus. Work on the quantitative relations be- 
tween amount of virus and production of Eous 
sarcoma in fowl led to greatly improved methods 
of extraction and concentration of this virus. At 
the same time a much more higUy susceptible host 
for murine tumors — the new-born mouse — was 
developed in work on the milk agent, the Gross 
leukemia, and polyoma viruses. It became possi- 
ble to grow polyoma virus in quantity on tissue 
culture of mouse kidney and mouse embryo cells. 
Concurrently, the refinements in the tecliniques of 
electron microscopy, especialy by one NCI scien- 
tist, has increased the ability to identify oncogenic 
virus particles. All of these techniques, beginning 
about 1957, were focused on the problem of find- 
ing new oncogenic viruses, with considerable 
success in the mouse leukemias. 

In 1958 the Moloney leukemia virus (MLV) 
was isolated from Sarcoma 37. Its history and 
characteristics have appeared in previous reports, 
and here only a few aspects will be considered. It 
should be emphasized that MLV has provided an 
excellent model to help understand the patho- 
genesis of virus-induced leukemia and to predict 
new relationships. 

The virus has been found budding from the 
plasma membrane of malignant lymphoblasts and 
the intracellular membranes of megakaryocytes. 
In the latter cells, a large number of virus par- 
ticles can be seen in the cytoplasm. They have 
also been demonstrated in platelets. The largest 
amount of virus, however, has been found free in 
the blood, and this provides a technique for isolat- 
ing large amomits of pure virus. The amounts 
to be harvested are limited by the size of the 
animal, and so far the hamster is the largest 
susceptible species. Attempts are under way to 
transmit the disease to monkeys and cattle, but 
these possibilities will not be fully examined until 
much larger doses of virus can be given to these 
larger animals. MLV is excreted in the milk and 
vertical transmission of the disease to suckling 
mice has been shown. Lateral transfer of virus 
lias not been found. 



AJSnsrUAL EEVIEW OF INTRAMURAL RESEARCH 



The pathologists have made good use of ]\ILV 
leukemia in mice and rats to observe early micro- 
scopic lesions. The first changes occur in thymus 
epithelium. This is followed by splenic hyper- 
plasia and later by lymphoid leukemic involve- 
ment of marrov? and lymph nodes. If the vinis 
is inoculated mto thymectomized mice, reticulum 
cell sarcoma results rather than leukemia. If 
inoculated into splenectomized mice myeloid 
(cliloro-) leukemia rather than lymphoid leukemia 
appears. However, prior to the appearance of 
microscopic evidence of leukemia in the rat, virus 
particles can be shown in megakaryocytes. 

MLV is now growing well in tissue cultures of a 
cell line derived from mouse spleen. The cell line, 
probably a reticular cell, is in continuous culture, 
and attempts are under way to increase the size of 
such cultures to produce large amounts of pure 
virus. 

The new techniques have resulted in the isola- 
tion of new leukemia viruses. Tliese ai'e the C-60 
virus isolated from a Swiss mouse with the 
Schoolman-Schwartz disease ; the E-]SrCI-2 virus 
from similar mice; and the Breyere-Moloney 
agent from Balb/c mice. 

The lessons of the murine leukemia viruses seem 
to hold some promise for clues to the solution of 
the clinical problem. Studies in both the animal 
and patient systems are proceeding, and based on 
murine leukemia it would seem higUy likely that 
a "virus" or some chemical or event capable of 
modifying the DISTA-ENA mechanisms would 
soon be identified in association with one of the 
human leukemias. In preparing for what at this 
moment seems inevitable, it is profitable to ask 
what should be provided for the scientist to facili- 
tate his examination of the various possibilities. 

Electron microscopy represents at this time the 
most rapid and direct end-point in relating bio- 
logical phenomena of tumors to the presence of a 
virus. But even this requires several months and 
a scientist can make relatively few observations, as 
shown by the fact that in 6 months only 16 of 100 
samples of human leukemic blood could be exam- 
ined. The problem is to insure the training of 
more men in this field. This is particularly true 
in Pathology and the clinic, for if a human tumor 
is found to be related to a virus or infectious 
nucleic acid then rapid examination must be avail- 
able for diagnosis. 



To find a more rapid way of proving the pres- 
ence of a virus other direct teclmiques are needed. 
The most promising at the moment is that of viral 
interference developed in the Laboratory of Viral 
Oncology. Based on the observation that Moloney 
virus will interfere with the action of influenza 
virus in fowl embryos, an assay is being developed 
which can be read in 5 days. However, the test 
is non-specific and a number of controls to rule out 
contaminating viruses are needed. Another solu- 
tion would be the rapid growth of the virus in 
tissue culture, but considerable research is needed 
on the cell lines themselves, as well as on the virus 
in culture, before any assay will be possible. 

Since the incubation period for the oncogenic 
viruses in annuals is in large part dependent on 
size of infective dose, considerable planning is 
mider way to provide facilities m which large 
amounts of viruses can be made available. An 
outside contract has been established which will 
not only ensure production of Moloney and 
Eauscher viruses in mice and rats but will also 
develop tissue culture as a source of large quan- 
tities of these viruses. Such large amounts of 
pure virus will speed up studies on assay systems, 
and will also permit examination of the chemical 
characteristics of the viruses, their transformation 
into infectious nucleic acids, the possibility of in- 
fecting large mammals such as primates, dogs, 
cattle, and horses, and the manufacture of vac- 
cines. 

Large amounts of the murine viruses will also 
permit the infection of large niunbers of animals 
for viral chemotherapy studies. Even with the 
amounts now available some of the chemotherapy 
studies in transplanted tumors have been replaced 
by studies of the chemotherapy of Moloney and 
Eauscher virus leukemias. Preliminary results 
indicate that the whole cell transmitted disease 
can be cured by means of drugs, but death ensues 
due to recrudescence of (Moloney) virus induced 
leukemia. Active collaboration with the Virus 
Eesearch Eesources Branch will provide chemicals 
with potential anti- viral activity. 

Pathogenesis and Bodily Economy 

In the animal given Moloney virus, particles are 
first demonstrated in the grossly nonnal megakar- 
yocyte and later in the malignant lymphoblast. 



NATIONAL CANCER INSTITUTE 



Then follows histologic change in the thymus and 
spleen, and finally, the production of excess lym- 
phoblasts which infiltrate many organs. Even if 
the same procession of events occurs in patients 
after the initiating leukemogenic stimulus, at the 
present time most observations in leukemic pa- 
tients are related to the late situation of leuhocj^te 
over-production with consequent replacement of 
normal tissues. The gxeatest impact m acute lym- 
phocytic leukemia is upon the bone marrow and 
the central nervous system. As a result of bone 
marrow infiltration there is interference with red 
cell production, and a sharp reduction m produc- 
tion of platelets and granulocytes, giving rise re- 
spectively to anemia, hemorrhage, and infection. 
If remission in the bone marrow phase of the dis- 
ease can be induced, there often occurs luxuriant 
growth of leukemic cells in the meninges with the 
production of hydrocephalus. Each of these ma- 
jor manifestations of acute lymphocytic leukemia 
has been studied in the clinic with the purpose in 
mind of controlling these life-threatening proc- 
esses. 

The anemias of the leukemias and related dis- 
eases have been examined by physicians of the 
Metabolism Section. The mechanism is not one 
of shnple failure of red cell synthesis due to bone 
marrow replacement. The widest spectrum of 
mechanisms was found in chronic myelocytic leu- 
kemia where one can demonstrate either an in- 
crease or decrease in rates of red cell production, 
and a shortened red cell life span. Later in the 
acute myeloblastic stage there is markedly short- 
ened red cell survival and an absence of red cell 
synthesis. It is difficult to generalize about the 
mechanism of anemia in a particular patient. As 
is true of so many of the physiologic deficits of 
disease, when precise methods of examination be- 
come perfected, the specific "anemia" can be shown 
to be the resultant of several processes, weighted 
differently and even oppositely, in several patients 
with the same disease. Wliile this heterogeneity. 
of mechanisms within single disease entity is dis- 
couraging for those who wish to generalize, their 
recognition sometimes permits specific corrective 
measures to be undertaken for a given patient. 

In addition to studies of the direct effects of 
leukocyte over-production upon the bodily econ- 
omy, there are nucleic acid abnormalities in some 
of the leukemias which may or may not be related 



to the excess white cells. Studies on nucleic acid 
biochemistry are being pm-sued intensively in a 
number of our laboratories. Most of these are 
necessarily in simple artificial systems; mention 
will be made here only of a few early attempts 
at studies m patients. 

In the Metabolism Section the main interest has 
been in pseudouridme (5-ribosyl-uracil) and 
uracil. Pseudouridme was found in some leukemic 
patients, and is of interest because it is not catabo- 
lized. Wlien labelled pseudouridine was used as 
a measure of pyrimidine production, it was calcu- 
lated that patients synthesized between 1.1 and 
1.6 grams of pyrimidine nucleoside per day. In 
the Laboratory of Physiology, pseudouridine has 
also been under study in patients and its excretion 
has been shown to be partially diet dependent. In- 
creased amounts of pseudouridine were found in 
acute lymphocytic leukemia but not m patients 
with acute myeloblastic type. 

The large number of closely related pyrimidiaes 
and their congeners in urine has held up progress 
in the study of their relationships to disease and 
metabolism. When data, processing equipment 
was put in the Laboratory of Physiology it became 
possible to resolve some of these problems, as weU 
as to make available to Institute scientists the 
possibility of fairly direct solution of other com- 
putational and data retrieval problems. The scien- 
tists of the Energy Metabolism Section have taken 
the leadership in assisting other scientists to pro- 
gram their problems and to develop mathematical 
models for the further exploration of their data. 
In^ addition to making feasible the analysis of 
urinary pyrimidines, programs have also been 
developed for the study of the sequences of bases 
in the nucleic acids; for the prediction of toxicity 
and therapeutic effect of drugs in the L1210 
screen; for retrieval and correlation of autopsy 
data ; and for the analysis of metabolic data. One 
can expect the rapid application of these types of 
programming to a variety of laboratory and 
clinical studies. 

Management of the Leukemias 

The ability of our physicians to influence favor- 
ably the course of patients with the acute leu- 
kemias has improved considerably in the past year. 
These improvements have resulted in better con- 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



trol of hemorrhage and infection, and of the disease 
itself by improved cliemotlierapy. 

The hemorrhagic complications of the acute 
leukemias have been reviewed by physicians of 
the Medicine Branch and have been shown to be 
due almost exclusively to lack of platelets. Stud- 
ies on platelet replacement have continued at a 
rapid pace, and it is now possible to use pooled 
platelets. This has permitted control of platelet 
level, and therefore hemorrhage, in adults as well 
as children, and at the present time 85% of 
patients with such difficulties can be satisfactorily 
managed. This has become a regailar ward pro- 
cedure, and although it has been done by the 
Institute, it is such a standard part of operations 
it will soon be transferred to the Blood Bank. In 
addition, research on preservation of platelets for 
future use should be midertaken in conjunction 
with the Blood Bank, since only when it becomes 
possible to store platelets in advance of need will 
full use of this procedure be made. 

The techniques learned in the gentle handling 
of platelets have led to successful studies of the 
replacement of granulocytes in the treatment of 
Pseudomonas septicemia. This is a common com- 
plication of acute leukemia and in a previous 
study was shown to be related to the absence of 
granulocytes. Physicians of the Medicine Branch 
have been able to give doses of 10-30 billion gran- 
ulocytes, obtained from patients with chronic 
granulocytic leukemia, to patients with Pseu- 
domonas septicemia who were also receiving anti- 
bacterial chemotherapy. In 22 patients with this 
infection treated in former years with chemother- 
apy alone, 21 were dead within 4 days and the 22nd 
survived about 10 days. Six of the last ten patients 
given granulocytes have been cured of their Pseu- 
domonas septicemia. Further work is proceeding 
in getting more granulocytes and obtaining them 
from normal donors. Future work must face up 
to the problems of granulocyte preservation; of 
the dangers of possible homotransplantation of the 
white cells, and the remote possibility of transfer 
of a "virus" of chronic granulocytic leukemia. 

In last year's report, attention was drawn to the 
usefulness of the L1210 mouse leukemia, as studied 
in the Laboratory of Chemical Pharmacology, to 
predict activity of new drugs for the acute leu- 
kemias. Using this model, two new agents of 
importance have been added — methyl-glyoxal- 



bis-guanylhydrazone (CH3-GAG) and the tere- 
phthalanilides. In addition, the model has pre- 
dicted a better way of using methotrexate but lias 
failed in picking up an agent vincristine (leuro- 
cristine) which is quite active in acute lympho- 
blastic leukemia. 

CH3-GAG has been used in 13 patients with 
acute granulocytic leukemia. Eleven have shown 
objective improvement and 9 of the patients have 
achieved complete remission. Since 6-MP, the 
only drug useful for this disease, induces less than 
20% complete remission, the new drug offers addi- 
tional benefit to the adult with acute leukemia. 
The drug has a low therapeutic index and is not 
absorbed after oral administration. A variety of 
analogues is being synthesized for additional 
studies. Its major toxic effect seems to be on 
the epithelium of the upper digestive tract. Tlie 
terephthalanilides, with high activity in L1210, 
have proven to give unexpected oculomotor pa- 
ralyses in man at doses below predicted therapeu- 
tic range. However, the L1210 model has recently 
shown that these drugs when administered with a 
small amount of methotrexate, give rise to a syn- 
ergistic effect. This observation is being extended 
to other anti-leukemia agents, and the methorex- 
ate-terephthalanilide combination is imder clinical 
trial. In addition, there are many analogues of 
the latter drug available for further screening 
for synergism. 

The L1210 model indicated sometime ago that 
methotresiate given every four days was more 
efficient than the same total amount of drug given 
by daily dosage. This has now been extended to 
patients with acute lymphocytic leukemia and 15 
of the first 18 patients (83%) treated intraven- 
ously are in complete remission. Since the daily 
dosage regimen of oral methotrexate gave only 
29% complete remissions, the comparison is en- 
couraging. A direct comparison in patients will 
now be made. 

Vincristine (leurocristine) is a drug developed 
by Eli Lilly and Company as an analogue of the 
periwinkle alkaloid, Vincaleukoblastin (Velban). 
It has marked activity in P-1534: leukemia, a lym- 
phocytic leukemia closely resembling L1210. Tlie 
activity in L1210 is only 20% of that of metlio- 
trexate. In 11 patients with acute lymphocytic 
leukemia, 7 have entered complete remission. In- 
terestingly, this was accomplished much more 



NATIONAL CANCER INSTITUTE 



rapidly than the antimetabolines and without bone 
marrow toxicity. It causes alopecia and some 
changes in peripheral nerve function. Other 
alkaloids of this series have been made available 
and -svill be studied. Also, there is under way a 
comparative study of the P-1534 and L1210 leu- 
kemias in order to study whether both are needed 
in the leukemia screen. 

With the above advances, namely, the ability 
to control hemorrhage and Pseudomonas infection, 
new drugs with a high remission rate in both forms 
of acute leukemia, a much more effective way of 
using methotrexate, and the possibility of a syner- 
gist for the antifolics, the survival rates and du- 
rations will be markedly increased. It is too soon 
to quantify the impact of these advances on sur- 
vival times, but one would expect that it would 
at least double in patients treated with all these 
techniques. How soon does one deliberately try to 
apply these new methods across the country? 
Part of tliis will be accomplished when the total 
survival figures are available and when the re- 
sults are published, but there is already sufficient 
proof to suggest that a more directed approach 
would result in a more prolonged life for those 
with acute leukemia. How this might be done by 
the joint efforts of the scientists of CCNSC, the 
intramural group and the cooperative groups is 
the subject of present planning. 



BIOCHEMICAL AIND PHYSIOLOGICAL 
STUDIES OF CLINICAL CANCER 

"Wliile the clinical branches of the National 
Cancer Institute are chiefly concerned with the 
diagnosis and treatment of cancer in man, a sig- 
nificant effort is being made within the National 
Cancer Institute in the study of the biochemistry 
and physiology of man as modified by the pres- 
ence of a tumor. They are studies intended to 
elucidate either mechanisms of disease, mecha- 
nisms by which the tiunor produces metabolic 
changes or studies in man of the biochemical and 
physiological problems associated with tlie treat- 
ment of cancer. These studies should be viewed 
as an integral part of the larger program. A 
review at this time of the activities in these fields 
of the National Cancer Institute would serve to 
indicate the broad approach that the clinical in- 



vestigators have developed for the study of dis- 
ease. In many instances parallel studies are being 
carried out in experimental animals. These will 
not be reviewed here since it is intended that this 
review be confined to studies in man. 

Nucleic Acids 

The nucleic acids, both DNA and RNA, are 
directive both qualitatively and quantitatively of 
macromolecular synthesis and thus are central to 
many metabolic processes. For this reason a 
number of studies of nucleic acid metabolism have 
been undertaken. 

Column chromatographic methods for isolating 
the pyrunidine pseudouridine (5 ribosyl-uracil) 
and uracil from urine have been developed. Ele- 
vated excretion of these compounds was foimd in 
patients with gout, leukemia, and psoriasis re- 
flecting increased catabolism of nucleic acids in 
leukemia and psoriasis, but in gout the explana- 
tion for this increased excretion is not at hand, 
since it is not correlated with uric acid excretion. 
The excretion diminished following administra- 
tion of azauridine because of suppression of 
uridine synthesis. Biosynthetically labeled pseu- 
douridme contaming C" or tritium was prepared 
in rats. In both man and the rats, pseudouridine 
is not catabolized; therefore, excretion rates re- 
flects synthesis rates in the steady state. Ring 
labeled orotic acid was administered to patients 
and the excretion of C^* as respiratory C^*02 
(greater than 50%) uracil (1-2 %) and pseu- 
douridine (2-3 %) determined. From the cumu- 
lative excretion of isotope as pseudouridine and 
its excretion rates, estimates of the pyrimidine 
production rate in three patients ranged between 
1.1 and 1.6 grams of pyrunidine nucleoside per 
day. These are the first estimates to be made of 
total pyrimidine synthesis in man. With C^* 
labeled uracil, evidence was obtamed indicating 
the virtual absence of re-utilization of this mate- 
rial, indicating that at nearly physiological levels, 
few cells of the body are likely to be able to use 
this compoimd as a substitute for endogenously 
produced pyrimidine. 

An unusual group of nucleotide-peptide com- 
pounds have been studied, certain of which were 
tentatively identified as 5' uridylic acid, adenylic 
acid, and 6-methyladenylic acid. The principle 



6 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



amino acids were glutamic acid and asparatic acid. 
Studies with P^- indicate a rapid turnover of tliese 
compounds. 

Elevation of serum uric acid has been fomid to 
be characteristic of psoriasis. Patients with this 
disease have been found to over incorporate C^* 
labeled glycine into uric acid as is also observed in 
primary gout, but to a lesser degree. This ap- 
pears to be secondary to an increased turnover rate 
of the hyperplastic psoriatic epidermis, since the 
degree of hyperuricemia and pseudouridine excre- 
tion are well correlated with the percentage of skin 
involved with psoriasis. 

Studies of the ribonucleic acid content of white 
blood cells derived from patients with chronic 
myelogenous leukemia and chronic lymphatic leu- 
kemia indicate that guanine and adenine exists in 
excess. There are also unusual adenylic acid like 
nucleotides present. These studies suggest that 
the white blood cell KISTA differs quantitatively 
from the ENA of more differentiated tissues. In 
chronic myelogenous leukemia and chronic lym- 
phatic leukemia, preliminary evidence indicates 
that acid soluble nucleotide containing compo- 
nents of ENA (phenol prepared) are relatively 
abundant as compared with those found in the 
liver. 

Adenylic, guanylic, and inosinic pyrophosphory- 
lase levels have been determined in leukocytes of 
patients with leukemia and in normal white blood 
cells. No consistent pattern of enzyme abnormal- 
ity has been observed. Patients who were clinic- 
ally resistent to G-mercaptopurine have not shown 
the loss of inosinic pyrophosphorylase activity that 
has been observed in various bacteria and in trans- 
plantable mouse tumor cells that develop resist- 
ance to 6-mercaptopurine. 

Azauridine, a pyrimidine analogue inhibits both 
in vivo and in vitro the enzyme, orotodylic decar- 
boxylase. This is one of the steps in pyrimidine 
synthesis. Administration of this compound to 
man results in excretion of orotic acid and oroti- 
dine. Since these two compounds occur in the 
metabolic process immediately prior to the in- 
hibited enzyme their appearance in the urine 
might be anticipated. Azauridine also induces a 
uricosuria and a lowering of the serum uric acid. 
This is thought to be a direct effect on the Iddney 
and represent inhibition of the tubular reabsorp- 



tion of uric acid. Tlie enzyme, orotodylic acid 
decarboxylase, m the white blood cells of patients 
with chronic myelogenous leukemia is depressed 
both in vivo after administration of the drug, and 
in vitro. Followmg therapy with this drug, par- 
tial remissions have been observed including: a de- 
crease in the white count and a decrease m organ 
size. In some patients after several weeks, the 
white blood count increases and the spleen en- 
larges in spite of continued azauridine administra- 
tion. Associated with this clinical evidence of 
resistance is a decrease in the degree of enzymatic 
inhibition. 

Amino Acids 

The urinary excretion of amino acids, as alpha 
amino nitrogen, in children with acute leukemia 
was found to be normal. 

Plasma Proteins 

Distinctive serum and urine proteins associated 
with multiple myeloma and certain lymphocytic 
neoplasms, characterized by inxraunochemical and 
physicochemical tecliniques have been found to fit 
into one of four classes of globulin : 6.6S gamma 
globulin. Beta 2a globulin, 18S macroglobulin or 
Bence Jones protein. By studies of electropho- 
retic, chromatographic, ultra-centrifugal proper- 
ties and antigenic determinants, the range of 
molecules formed in malignant plasma cells was 
defined, and a comparison of these proteins with 
those in normal servim undertaken. The proteins 
formed in malignant plasma cells closely resem- 
bled normal globulins and, in fact, could not be 
shown to be abnonnal either in man or the mouse. 

The genetic properties of malignant plasma 
cells were assessed by measurement of the Gm 
groups a, b, and x present on myeloma proteins 
and macroglobulins. Normal gamma globulins 
have these genetic traits which reflect the genetic 
constitution of normal plasma cells. The 6.6S 
gamma myeloma proteins also were found to carry 
the Gm genetic traits indicating that the genetic 
properties of malignant plasma cell (in which 
myeloma proteins are formed) are similar to the 
genetic properties of normal plasma cells. 

Ultrafiltration using nitrogen under high jares- 
sure and a collodion membrane for the concentra- 
tion of dilute solutions containing proteins of 



NATIONAL CANCER INSTITUTE 



dilute solutions containing proteins of cerebro- 
spinal fluid, sweat, and urine have been carried out. 
These concentrated solutions were then studied 
by paper electrophoresis. Abnormal gamma glob- 
ulins, Beta 2a and Beta 2m globulins were present 
in the nasal secretions in patients with multiple 
myeloma. The cerebro-spinal fluid in central 
nervous system leukemia shows minor changes in 
the albumin and Beta globulin components. 
There was no protein found in the sweat in pa- 
tients with multiple myeloma. This method of 
concentration of proteins also proved satisfactory 
for studying protein excretion in the urine. 

Twenty patients with lyniphomatous disease 
were studied with Iodine 131 labeled albumin to 
determine the rate of synthesis of albumin, total 
body albumin, and if the gastrointestinal tract was 
a site of protem loss in these patients. These in- 
cluded three patients with marked diarrhea and 
three with large chylous effusions. Ten of these 
twenty patients, and an additional ten, were also 
studied using iodinated polyvinylpyroUidon 
( P VP ) . All but five of these patients had a serum 
albumin concentration \mder 3.5 grams percent. 
The three patients with chylous effusions had a 
prolonged albimiin survival and an increased total 
albumin pool. The remaining 17 patients had 
markedly reduced body albumin pools with a nor- 
mal or a slightly short albumin survival, but de- 
creased albumin synthesis. In no instance in a 
patient in this group was gastrointestinal protein 
loss demonstrated. 

The preparation of chromium 51 labeled albu- 
min and the demonstration that it was superior to 
iodinated PVP represents a major advance in the 
means for detectmg gastrointestinal protein loss. 

Biochemistry of the Skin 

Protein fractions have been extracted from ex- 
foliated scales of psoriasis. Both soluble and in- 
soluble fractions have been found which can then 
be further fractionated into four sub-fractions. 
Following the administration of glycine C^*, gly- 
cine was isolated from each of these eight fractions 
and its radioactivity determined. The time of ap- 
pearance of isotope in each of these fractions dif- 
rered, indicating that each fraction was synthe- 
sized at a different rate within tlie epidermis or 



at a different time foUowmg administration of 
the isotope. 

Split thickness skin grafts cultured in a medium 
contaming tritiated thymidine showed evidence of 
synthesis of DNA. This important observation 
indicates that imder the conditions used, the skin 
continues to be capable of synthesis and opens the 
way to many further studies of the biochemistry 
of skin. 

Incubation of the skin of subjects with albinism 
and vitiligo with tyrosine indicated the presence 
of tyrosmase in the skin of the albino, but not in 
the depigmented skin of persons with vitiligo. 
Thus the pigment synthesizing defect in albinism 
is still to be determined. 

Endocrine Studies 

Patients with multiple basal cell carcinoma have 
been given parathormone and their urinary phos- 
phate excretion was measured. Two patients with 
the syndrome of basal cell carcinoma, bifid ribs 
and cysts of the mandible have failed to respond 
to this hormone with phosphate diuresis as did 
patients with ordinary basal cell carcinoma and 
five normal subjects. 

Seven of 95 patients with trophoblastic disease 
were hyperthyroid as evidenced by elevated PBI 
and/or P^^ uptake. In several of these patients, 
the hyperthyroidism or apparent hyperthyroidism 
subsided after treatment with methotrexate which 
led to substantial tumor regression. 

Study by the Ouchterlony diffusion technique 
and immimoelectrophoresis indicates that the 
chorionic gonadotropic hormone in pregnancy is 
associated with a gamma globulin, whereas in 
choriocarcinoma it is f oimd with the beta globulin 
fraction. Antisera produced to both gonadotro- 
phins were shown to be equally potent indicating 
that from the immunological standpoint tliese ma- 
terials are equivalent. 

Section of the pituitary stalk in patients with 
advanced breast cancer shows that thyi-oid func- 
tion may persist after interruption of the hypo- 
thalmic hypophyseal porial system indicating that 
the thyroid, in some instances, functions inde- 
pendently of the hypothalamus. 

In collaboration with Dr. B. Hokfelt of the 
Karolinska Hospital, it has been shown that 17 
alpha-hydroxy pregnenolone is a precursor of cor- 



8 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tisol in man as it is in the rat and the guinea pig. 
This is a previously unidentified pathway of Cor- 
tisol synthesis in the adrenal gland. Testosterone 
was synthesized from progesterone in slices from 
the ovarian cyst wall taken from a patient with 
a virilizing syndrome. Measurement of aldoster- 
one excretion and secretion rates indicate that the 
diuresis that occurs in patients with refractory 
edema can occur without change in aldosterone 
excretion rates. 

Patients with adrenal cancer treated with 
o,p'DDD absorb only 20% of an oral dose. Wlien 
the body fat stores become saturated an approxi- 
mately constant blood level can be maintained. 

Studies on patients with adrenal cortical car- 
cinoma have shown that in those patients with 
Cushing's syndrome, an increased excretion of 
tetrahydro S substances has been found in each 
patient; whereas the increased excretion of de- 
hydro-epiandiosterone is not constant. In patients 
with adrenal cortical carcinoma isotope dilution 
studies demonstrated that the increased etiochol- 
analone : androsterone ratio resulted from the me- 
tabolism of C^^ precursors in contradistinction to 
findings in the normal subject. 

Hematopoiesis 

The effects of A^arious doses and schedules of 
endotoxin on fever, WBC changes and antibody 
production was studied in normal prisoner volun- 
teers. Differing dose-response relationships ob- 
tained for fever index, mature granulocyte incre- 
ment and immature granulocyte increment. The 
variability of response to a given dose was slight. 
In patients with neoplastic disorders, the variabil- 
ity of response to a given dose was greater and 
appeared to be related to involvement of the 
reticuloendothelial system. With repeated daily 
endotoxin administration tolerance to the febrile 
effect occurred within 7 days whereas the granu- 
locyte increment persisted. 

Studies of hematopoiesis in patients undergoing 
total body radiation indicates that the increase 
in the half time for the plasma clearance of intra- 
venously administered radioiron precedes any 
change in hemoglobin, reticulocyte, or platelet 
counts, and furthermore, that the clearance half 
time returns to normal before the reticulocyte 



count and hemoglobin rises. Post-radiation, white 
blood cell mobilization in response to endotoxin is 
progx'essively depressed. 

A new assay system for erythropoietin, using 
tlie polycythemic mouse has been developed. It 
exhibits markedly increased sensitivity and re- 
quires smaller volumes of starting materials than 
previously available procedures. Erythropoietin 
has been demonstrated in an extract of cerebellar 
hemangioblastoma tumor tissue, cerebellar cyst 
fluid and the plasma of a patient with poly- 
cythemia, but not in the renal cyst, pancreatic 
cyst fluid, or an extract of a kidney tumor from 
this patient. Erythropoietin has also been dem- 
onstrated in an extract of the tumor from a 
patient with pheochromocytoma who was poly- 
cythemic, and from the plasma of two of three 
additional patients with pheochromocytoma. 
These findings suggest that the polycythemia as- 
sociated with pheochromocytoma and cerebellar 
hemangioblastoma is a result of production by 
these tumors of an erythropoietic substance. In 
five of ten patients with renal cystic disease ery- 
throiDoietin has been demonstrated in the renal 
cyst fluid. Three of the five patients were poly- 
cythemic. In two of three cases, resection of the 
renal cyst produced a reversal of the polyc)'- 
themia. These studies suggest that the kidney 
is a major site of erythropoietin synthesis in 
man. Elevated erythropoietin titers have been 
demonstrated in the serum of only one of four 
patients with polycythemia secondary to von Hip- 
pel Lindau's disease and in none of the six pa- 
tients with polycythemia vera. A group of 
patients with leukemia, multiple mj^eloma, chronic 
infection and aplastic anemia liave been found 
to have elevated erythropoietin levels when the 
peripheral hemoglobin concentration was less 
than 8 grams percent even when there was severe 
renal impairment. This indicates that the anemia 
of renal disease is not due to the absence of ery- 
thropoietin. There was a direct correlation of 
the erythropoietin titer with the severity of the 
anemia. With the exception of tlie patients with 
aplasia of the bone marrow, there was little or no 
correlation between the erythropoietin level and 
the reticulocyte count. Endogenous ei-ythropoi- 
etin production was decreased by transfusing 3 
anemic subjects with three units of packed cells. 



NATIONAL CANCER INSTITUTE 



9 



The plasma erythropoietin activity decreased 
with half times of 9% to 19 hours. 

The molecular weight of the biologically active 
portion of erythropoietin was found to be 25- 
30,000 using varying doses of radiation from the 
van de Graff accelerator for inactivation and 
assuming a spherical molecular shape. 

Diisoproplyfluorophosphate P^^ (DFP^^) was 
evaluated as a label for the measurement of red 
cell life span in the dog and man, and found to 
be satisfactory; in fact, probably the method of 
choice for most situations requiring this measure- 
ment. 

Studies of erythropoiesis including determina- 
tion of total red cell volume, rate of production 
of red cells and red cell life span were carried 
out in patients with multiple myeloma, macro- 
globulinemia, Hodgkin's disease and chronic mye- 
locytic leukemia. In multiple myeloma, the 
predominant pattern of erythropoiesis was that 
of erythropoietic insuiEciency with a moderate 
shortening of red cell survival. The red cell life 
span was finite but short. Variations in these 
patterns were found depending upon the stage of 
disease. Androgen therapy in large doses pro- 
duced an increase in the rate of production of 
red cells and an increase m the total red cell vol- 
ume in four of five patients studied. In chronic 
myelocytic leukemia a spectrum of patterns of 
erythropoiesis emerged. This varied from a nor- 
mal total red cell volume, normal rate of pro- 
duction of red cells, and normal red cell life span 
through increased to decreased rate of produc- 
tion of red cells; a finite shortening of red cell 
life span in the order of 75 to 80 days, and later 
markedly decreased erythrocyte survival in tlie 
acute myeloblastic stage with a virtual absence 
of red cell synthesis. In macroglobulinemia, sev- 
eral distinct patterns were found. At one ex- 
treme, two patients with erythroid aplasia wei-e 
observed, and at the other extreme, marked short- 
enmg of the red cell life span. In two patients, ■ 
shortening of red cell life span could be related 
to clinical activity of the disease and the concen- 
tration of serum macroglobulins. A preliminary 
review of studies in nine patients with Hodgin's 
disease indicates that shortening of red cell life 
span is the predominant abnormality except in 
the late stage where there is bone marrow failure. 



Folic Acid Antagonists 

Dicliloromethotrexate (DCM) containing ra- 
dioactive chlorine was given to patients both 
intravenously and orally to study the mechanisms 
involved in the metabolism of this compound in 
an attempt to explain the differences in thera- 
peutic efficacy in the mouse as compared to man. 
Following oral administration the urinary out- 
put of isotope was less than when given intra- 
venously. This is thought to be due either to 
a failure to absorb a part of the dose through the 
gastrointestinal tract or alternatively that follow- 
ing absorption from the gastrointestinal tract the 
material is transported directly to the liver where 
it is excreted into the bile. The urinary excre- 
tion pattern shows an increasing percentage of 
the isotope being excreted as a metabolite, dj'- 
hydroxy-dichloromethotrexate. This metabolic 
pattern is similar to that observed in the mouse. 
This differs from studies of the same compound 
in the dog in which animal this material is not 
metabolized, and in the rat in which the material 
is converted to the matabolite at a higher rate. 
These studies have been made possible by signifi- 
cant advances in the development of methods for 
a separation of folic acid-like compounds on 
DEAE columns and by high voltage electro- 
phoresis. 

Metabolic Balance Technique 

A common metabolic technique in the experi- 
mental animal is paired feeding in which the ex- 
perimental animal receives a diet, or is allowed to 
select a diet ad Itbitmn and a second animal is fed 
an identical diet. This permits an evaluation of 
the effect of any procedure by comparison with a 
laormal animal receiving an identical diet. A 
somewhat modified version of this technique con- 
sists in the preparing of identical trays of food 
for each meal and allowing the patient to select 
food ad libitum. Then by analyzing the initial 
diet and compai'ison with the residue left, the total 
intake is calculated. In this manner the effect of 
applied variables on the selection of food both in 
terms of quantity, chemical composition and bal- 
ance can be evaluated. This technique has been 
successfully applied and data concerning the 
anabolic and catabolic effects of tri-iodo thyronine. 



10 



ANNXTAL REVIEW OF INTRAMURAL RESEARCH 



cortisone, testosterone, and growth hormone have 
been readily demonstrated. Little or no imme- 
diate quantitative or qualitative effect on food 
intake was observed in conjunction with profound 
anabolic and catabolic stimuli. 



Calcium Metabolism 

Calcium metabolism was studied in patients with 
multiple myeloma with lytic bone lesions and in 
patients with breast cancer metastatic to the skele- 
ton. With radioactive calcium.*'' and metabolic 
balance techniques estimates of the rate of bone 
formation, bone destruction intestinal absorption 
and endogenous calcimn secretion have been car- 
ried out. All patients studied have been in nega- 
tive calcium balance particularly as a result of a 
markedly increased intestinal excretion rather 
than excessive urinary loss or inadequate absorp- 
tion from the gastrointestinal tract. External 
counting over bone revealed that the osteoblastic 
metastases of breast carcinoma pick up labeled 
calcium in greater quantities than does normal 
bone, while the lytic lesions of multiple myeloma 
show little deviation from normal. 



Serum Lactic Dehydrogenase 

A method for the determination of serum lactic 
dehydrogenase has been set up and values have 
been established for the normal. In various dis- 
ease states when disease is clinically static the 
"\^alues obtained are stable indicating that tech- 
nically the procedure is satisfactory. Following 
total body radiation of patients with chronic 
leukemia, within approximately one hour, the se- 
rum lactic dehydrogenase level falls reaching a 
minimum at 24 to 48 hours. This represents one 
of the earliest measurable changes following radia- 
tion. In patients with solid tumors, particularly 
cervical epidermoid carcinoma and adenocar- 
cinoma, the serum lactic dehydrogenese levels are 
variable from case to case and at the moment can- 
not be correlated Avith any other measurement of 
disease state. Three patients with rhabdomyosar- 
coma had consistent and elevated levels. Follow- 
ing extensive surgical procedures, the rise in serum 
lactic dehydrogenase was not comparable to that 
seen following a myocardial infarct; although in 
the surgical procedure considerably more tissue 



may be traiunatized. In Hand-SchuUer-Christian 
disease, elevated sermn lactic dehydrogenase levels 
have been found. 



Mitosis Inhibiting Activity in the Serum 

Serum from patients with cancer and other dis- 
eases and from normal controls has been extracted 
with ether and saline and the fractions injected 
into partially hepatectomized rats. In the normal, 
two fractions are found. One that inhibits he- 
patic cell regeneration, and the second, that stimu- 
lates hepatic cell regeneration. The serum from 
patients with cancer lack tliis inhibiting fraction. 
This deficit can be replaced with the inhibiting 
fraction. Administration of the inhibiting frac- 
tion delays the time of appearance and slows the 
rate of growth of the L-1210 mouse leukemia. 

Carcinoid 

The excretion of 5-hydroxy indolacetic acid and 
total indole excretion were decreased in patients 
given Cytoxan and 5-fluorouracil ; however, when 
these patients were calorically restricted to the 
same extent as occurred during drug therapy, the 
same effect was obtained, thus no effect of the 
drug per se could be demonstrated. This is m con- 
trast to similar studies in mice with a mast cell 
tiunor. 



Immunology 

Studies of the effect of malignant disease on 
immime processes were undertaken in patients 
with multiple myeloma and with macroglobuli- 
nemia. The serum levels of the gamma globulins 
and related immunoglobulins were low in most of 
the 40 myeloma sera and 16 macroglobulinemia 
sera tested, indicating that serum antibody levels 
might be low in these malignant diseases. Tliis 
was supported by the finding of low isohemag- 
glutinin levels in multiple myeloma (35 of 40 
patients) and macroglobulinemia (all of nine 
patients) . Antibody response to antigen adminis- 
tration, now being tested, appears to be deficient 
in these two diseases. 

The molecular basis of immunity has been under 
investigation by both physiochemical and immuno- 
chemical techniques. Previous studies have shown 



NATIONAL CANCER INSTITUTE 



11 



that antibodies in man may be among the 6.6S 
gamma globulins or the 18S macroglobiilins, and 
studies elsewhere have indicated that Beta 2a 
globulins may carry antibody activity. These 
three groups of proteins have distinctive properties 
but they also share some properties in common. 
This possibility that antibody-forming cells have 
some specific common property was investigated 
through structural studies of these three classes 
of globulins. Enzymatic fragmentation of gamma 
and Beta 2a globulins provided evidence that these 
proteins are made up of three subunits, and that 
two of the subunits in each class (gamma or Beta 
2a) were similar, whereas the third subimit was 
markedly different for gamma and Beta 2a globu- 
lins. Such studies narrow the molecular compo- 
nents in which distinctive globulin properties are 
sought, and help to put immunologic observations 
on a physiochemical basis. 



METABOLISM SERVICE 

Amino Acid Transport 

In vitro studies using C" and S^^ labeled amino 
acids in rat renal cortex slices indicate that this 
tissue accumulates amino acids in intracellular 
water from two to five times the concentration in 
extracellular fluid. This fulfills the major crite- 
rion of an active transport system. Using Mich- 
aelis-Menten enzyme kinetic methods, an affinity 
constant was calculated for each amino acid 
studied. Compartmental analysis of the data has 
resulted in a formulation of a three compartment 
transport model from which influx and efflux rate 
constants have been calculated. Plilorizin was 
found to stimulate cellular accumulation of amino 
acids although it is a potent inhibitor of the trans- 
port of sugar. This was shown to result from 
specific inhibition of the efflux of amino acids 
without demonstrable influx effect. Lysine, orni- 
thine, arginine, and cystine transport studies were ' 
undertaken in search of a common carrier mech- 
anism since all four are excreted in excess in hu- 
man cystinuria. Arginine and ornithine inhibited 
the transport of lysine C" due to competitive 
inhibition. 

Eats fed either a 30% galactose diet or maleic 
acid develoji amino-aciduria which results from 
defective renal tubular absorption. In vitro 



studies with maleic acid suggest that inhibits both 
influx and efflux. These studies are part of a pro- 
gram defining the mechanisms of amino acid trans- 
port and determining the relationship between 
transport and subsequent protein synthesis. 

Plasma Proteins 

The study of plasma proteins on the Metabolism 
Service has been concerned with: (1) The physi- 
cal-chemical and immunochemical properties of 
proteins pi'oduced in normal and malignant 
plasma cells, and, (2) Metabolic behavior in terms 
of rate of synthesis and degradation. 

Proteins in normal and malignant plasma cells 

The distinctive serum and urine proteins asso- 
ciated with multiple myeloma and certain 
lymphocytic neoplasms were characterized by 
immunochemical and physicochemical techniques 
and found to fit into one of four classes of globu- 
lin : 6 :6S gamma-globulin. Beta 2a globulin, ISS 
macroglobulin or Bence Jones protein. Parallel 
studies of sei-um and urine proteins associated with 
20 transplantable plasma cell tumors in mice estab- 
lished the existence gamma and Beta 2a myeloma 
j)roteins and Bence Jones proteins in this species 
which are vei-y similar to the analogous protems 
of man. By detailed studies of electrophoretic 
chromatogTaphic and ultracentrifugal properties 
and antigenic determinants, the range of molecules 
formed in malignant plasma cells was defined, and 
a comparison of these proteins with those in nor- 
mal serum coiUd be undertaken. The proteins 
formed in malnignant plasma cells closely resem- 
bled nonnal globulins and, in fact, could not be 
shown to be abnormal either in man or the mouse. 

Biosynthetic studies with plasma cell tumor 
slices and C^* labelled amino acids showed clearly 
that Beta 2a myeloma proteins and Bence Jones 
proteins as well as gamma myeloma proteins are 
synthesized in plasma cell tumors. Thus, the 
properties of these proteins reflect the properties 
of the malignant plasma cell. 

The genetic properties of malignant ]^lasma cells 
were assessed by measurement of the Gm groups 
a, b and x present on myeloma proteins and 
macroglobulms. ISTormal gamma globulins have 
these genetic traits which reflect the genetic con- 
stitution of normal plasma cells. The 6.6S gamma 



12 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



myeloma proteins also were found to carry tlie 
Gm genetic traits indicating that the genetic prop- 
erties of malignant plasma cells (in which mye- 
loma proteins are formed) are similar to the ge- 
netic properties of normal plasma cells. 

Metabolic Behavior 

These studies are principally concerned with 
measurement of the turnover of plasma proteins. 
Transplantable plasma cell tumors in mice acceler- 
ate the rate of turnover of gamma globulins and 
gamma myeloma proteins without altering al- 
bumin metabolism. Plasma cell tumors, however, 
differ in their metabolic effects for a plasma cell 
tumor producing Beta 2a globulins did not ac- 
celerate gamma globulin turnover. The gamma 
globulins formed in malignant plasma cells were 
found to behave metabolically in the same way as 
proteins produced in normal plasma cells. Gamma 
myeloma proteins were foiind to be synthesized at 
the same rate per gram of tissue origin as albumin. 

Twenty patients with lymphomatous disease 
were studied with Iodine 131 labeled albumin. 
These included three patients with marked diar- 
rhea and three with large chylous effusions. Ten 
of these twenty patients, and an additional ten, 
were also studied using lodinated polyvinylpyrol- 
lidon (PVP). All but five of these patients had 
a serum albumin concentration under 3.5 grams 
percent. The three patients with chylous effu- 
sions had a prolonged albumin survival and an in- 
creased total albmnin pool. The remaining 17 
patients had markedly reduced body albumin 
pools with a normal or a slightly short albumin 
survival, but decreased albumin synthesis. In no 
instance in a patient in this group was gastro- 
intestinal protein loss demonstrated. In four pa- 
tients with Wliipple's disease marked gastrointes- 
tinal protein loss was demonstrated. lodinated 
albumin turnovers on two of these patients showed 
a shortened albumin survival. These studies indi- 
cate that deficient protein synthesis is also a factor 
in hypoproteinemia seen in patients with Whip- 
ple's disease. "Wlien treated with antibiotics and 
steroids there was almost a complete reversal of 
this gastrointestinal protein loss. Twenty-seven 
patients with idiopatliic hypoproteinemia with 
serum albumin concentration under 2.5 grams per- 
cent were studied. None of these patients had 



renal or hepatic disease. Twenty-five patients had 
idiopathic hypocatabolic-hypoproteinemia. The 
synthetic rate for albiunin was normal or increased 
in all patients but gastrointestinal protein loss 
resulted in a shortened albumin survival. The 
features that these twenty-five patients shared in 
common was as follows: they were all below the 
age of twenty-eight at onset, had generalized 
edema, redviced seriun albumin, total globulin, 
gamma globulin, and iron binding protein. There 
was a normal or low sermn cholesterol. The major- 
ity of the patients had only mild gastrointestinal 
symptoms. Within this group of 25 patients, four 
clinical syndromes could be recognized. The first 
consisted of three patients with transient disease, 
which disappeared spontaneously in six weeks to 
three months. The second group included four 
patients with cardiac lesions, three with constric- 
tive pericarditis and an interatrial septal defect in 
one. Surgical correction of the cardiac lesions 
produced a reversal of the protein abnormalities. 
Three patients had chronic pulmonary disease, 
eosinophilia and a familial history of allergy. In 
all three marked amelioration of the gastrointes- 
tinal protein loss followed therapy with adreno- 
cortical steroids. In 15 patients gastrointestinal 
protein loss was thought to be due to dilated lym- 
phatic channels in the small intestine. Micro- 
scopic examination of the small bowel in these 
patients showed edema of the wall and a dark 
brown pigmentation. The lymphatics were di- 
lated and contained lipomacrophages. The mesen- 
teric lymphatics were greatly thickened and frag- 
mented. Surgical, steroid, and dietary treatment 
of these patients was ineffectual. 

Twenty patients with hypogammaglobulinemia 
were studied. Fifteen patients had serum albumin 
concentrations under 3.5 grams percent and in six 
there was less than 2 grams percent. Three of 
these six patients had decreased albimiin synthesis 
while in the remaining three, gastrointestinal 
protein loss was the major factor in producing 
hypoalbuminemia. This protein loss was reversed 
on therapy with cortisone or tetracycline, and a 
gluten free diet. 

Two patients with failure to produce albumin 
were studied. In both, the lodinated gamma glob- 
ulin survival was normal, indicating that the 
catabolism of albumin and gamma globulin are 
independent. All the abnormalities in these pa- 



NATIONAL, CANCER INSTITUTE 



13 



tients could be corrected by albumin infusion with 
tlie exception of a prolonged albumin survival. 

The demonstration that chromium 51 labeled 
albumin was superior to iodinated PVP repre- 
sents a major advance in the means for detecting 
gastrointestinal protein loss. 

Immunologic Studies 

Studies of tlie effect of malignant disease on 
immune processes were undertaken in patients 
with multiple myeloma and with macroglobuline- 
mia, and two transplantable plasma cell tumors 
in mice. The serum levels of the gamma globu- 
lins and related immunoglobulins were low in 
most of the 40 myeloma sera and 16 macroglob- 
ulinemia sera tested by immunoelectrophoretic 
and chromatograpliic tecliniques, indicating that 
serum antibody levels might be low in these malig- 
nant diseases. This view was supported by the 
finding of low isohemagglutinin levels in multiple 
myeloma (35 of 40 patients) and macroglobu- 
linemia (all of nine patients) . Antibody response 
to antigen administration is now being tested and 
appears to be deficient in these two diseases. 

When challenged witli specific antigens mice 
bearing transplantable plasma cell tiunors had 
impaired antibody production. Tlie two tumors 
differed, however, in their quantitative effects, in- 
dicating tlie need for furtlier investigation on the 
mechanisms of immunity. For tliis purpose and 
to establish a laboratory model for detailed inves- 
tigation of tumor effects on immunity, investiga- 
tions of the characteristics of antibody production 
and the properties of antibodies were undertaken 
in imbred mice. Studies to date clearly indicate 
that mice, lilve man, can produce both 6.6S gamma 
globulin and 18S niacroglobulin antibodies. 

The molecular basis of immunity has been under 
investigation by both physiochemical and immu- 
nocliemical techniques. Previous studies have 
shown that antibodies in man may be among the 
6.6S gamma globulins or tlie 18S macroglobulins, 
and studies elsewhere liave indicated that Beta 2a 
globulins may carry antibody activity. These 
three groups of proteins have distinctive proper- 
ties but they also share some properties in com- 
mon. This possibility that antibody-forming cells 
have some specific common property was investi- 
gated through structural studies of these three 



classes of globulins. Enzymatic fragmentation of 
gamma and Beta 2a globulins provided evidence 
that these proteins are made up of three subunits, 
and that two of the subunits in each class (gamma 
or Beta 2a) were similar, whereas the third sub- 
unit M'as markedly different for gamma and Beta 
2a globulins. Such studies narrow the molecular 
components in which distinctive globulin proper- 
ties are sought, and help to put immunologic ob- 
servations on a physiochemical basis. In addition, 
the functional and genetic properties of malignant 
plasma cells can be compared with normal plasma 
cells by means of the proteins (gamma globulins) 
produced in these cells. Related studies with my- 
eloma proteins are described in section on Plasma 
Proteins. 



Nucleic Acids 

Column chromatographic methods for isolating 
pseudouridine and uracil from urine were devel- 
oped. Elevated excretion of these compounds was 
found in patients with gout, leukemia, and 
psoriasis. The excretion diminished following ad- 
ministration of azauridine. Biosynthetically 
labeled uridine containing C ^* and tritium was 
prepared. In both man and the rat, pseudouridine 
is not metabolized. Ring labeled orotic acid was 
administered to patients and the excretion of C ^*, 
as respiratoiy C^*02, uracil and pseudouridine 
followed. From these data, estimates of the py- 
rimidine production rate in three patients ranged 
between 1.1 and 1.6 grams of pyrimidine nucleo- 
side per day. These are the first estimates to be 
made of pyrimidine synthesis rate in man. With 
C ^* labeled uracil evidence was obtained indicating 
the virtiial absence of re-utilization of this 
material. 

An imusual group of nucleotide-iDeptide com- 
pounds have been studied, certain of which were 
tentatively identified as 5' adenylic acid, 5' uridylic 
acid, adenosine diphosphate and 6-methyladenylic 
acid. The principle amino acids were glutamic 
acid and aspartic acid. Studies with P ^^ indicate 
a rapid turnover of these compounds. 

Calcium Metabolism 

Calcium metabolism was studied in patients 
with multiple myeloma with lytic bone lesions or 



14 



ANlSrUAL REVIEW OF INTRAMURAL RESEARCH 



ill patients with breast cancer metastatic to the 
skeleton. With radioactive calcium 47 and meta- 
bolic balance teclmique estimates of the rate of 
bone formation, bone destruction, intestinal ab- 
sorption and endogenous calcimn secretion have 
been carried out. All patients studied have been 
in negative calcium balance particularly as a re- 
sult of a markedly increased intestmal excretion 
rather than excessive urinary loss or inadequate 
absorption from the gastrointestinal tract. Ex- 
ternal countmg over bone revealed that the oste- 
oblastic metastases pick up labeled calcium in 
greater quantities than does normal bone, while 
the lytic lesions of multiple myeloma show little 
deviation from normal. 

Porphyrin Metabolism 

Studies of the biochemical lesions of porphyria 
in patients and in experimental animals have been 
carried out. In patients with acute intermittent 
porphyria an increase m carbohydrate and/or 
protein intake decreases the rate of excretion of 
porphobilinogen. Orthostatic hypotension and 
the "restless leg" syndrome have been demon- 
strated as manifestations of acute porphyria. An 
elevation of the protein bound iodine was found; 
this may be related to a diminished rate of deg- 
radation of the thyroid hormone. 

The synthesis of delta-aminolevulinic acid, the 
porphobilinogen was studied in the liver in vitro. 
There appears to be diminished ability to oxidize 
the alpha carbon atom of glycine to CO2 in ex- 
perimental porphyria. In experimental porphyria 
the foUowmg studies of the rate of utilization or 
oxidation were normal: rate of utilization of 
porphobilinogen and the rate of oxidation of the 
four carbon atom of delta-aminolevulinic acid, the 
oxidation of alpha-ketoglutarate, the one and six 
carbon atoms of glucose, the two carbon atom of 
pyruvate and the two carbon atom of acetate. 

Erythropoiesis 

The studies of erythropoiesis can be divided into 
the following areas: (1) The studies on site pro- 
duction of erythropoietin, (2) Studies on means 
of measuring red cell survival, (3) Clinical studies 
of erythropoiesis. 



Erythropoietin Production 

Paraboitic rats in which one partner of the pair 
was either nephrectomized or the ureter ligated 
and exposing one ainmal to room air, and the other 
to a mixture of 9% oxygen indicates that the 
kidney is the major but not the sole site of erythro- 
poietin production. A new assay system for 
erythropoietin, usmg the polycythemic mouse of 
markedly increased sensitivity and requiring 
smaller volumes of starting materials has been 
developed. This assay system utilizes the polycy- 
themic mouse. 

Erythropoietin has been demonstrated in an 
extract of cerebellar hemangioblastoma tumor 
tissue and cerebellar cyst fluid, the plasma of a 
patient with polycythemia and von Hippel Lin- 
dau's diseases but not in the renal cyst, pancreatic 
cyst fluid, or an extract of a kidney tmnor from 
this patient. Erytlu-opoietin has also been dem- 
onstrated in an extract from the plasma of two 
or three additional patients with pheochromocy- 
toma. These iuidings suggest that the poly- 
cythemia associated with pheochromocytoma and 
cerebellar hemangioblastoma is a result of produc- 
tion by these tumors of an eiythropoietin. Eenal 
cyst fluid and/or plasma has been obtained from 
ten patients with renal cystic disease and in five of 
ten patients with renal cystic disease erythropoie- 
tin has been demonstrated in the cyst fluid. Three 
of the five patients were polycythemic. In two of 
three cases, resection of the renal cyst produced a 
reversal of erythropoietin production in man. 
Elevated erytliropoientin titers have been demon- 
strated in the serum of only one of four patients 
with polycythemia secondaiy to von Hippel 
Lindau's disease and in none of the six patients 
with polycythemia vera. A group of patients 
with leukemia, multiple myeloma, chronic hifec- 
tion and aplastic anemia have had elevated eryth- 
ropoietin levels when the peripheral hemoglobin 
concentration was less than 8 grams even when 
there was severe renal impairment. Tliis indi- 
cates that the anemia of renal disease is not due 
to the absence of erythropoietin. There was a 
direct correlation of the erythropoietin titer with 
the severity of the anemia. With the exception 
of the patients with aplasia of the bone marrow, 
there was little or no correlation between the 
erythropoietin level and tlie reticulocyte count. 



NATIONAL CANCER INSTITUTE 



15 



Endogenous erythropoietin production was de- 
creased by transfusing 3 anemic subjects with three 
units of packed cells. The erythropoietin activity 
decreased with a half time of Qi/^ to 19 hours. 

The molecular weight of the biologically active 
portion of erythropoietin was found to be 25- 
30,000 using varying doses of radiation from the 
van de Graif accelerator and assuming a spherical 
molecular shape. Using the polycythemic mouse 
it was determined that the control of erythropoie- 
sis is determined by the total red cell volume rather 
than the peripheral hematocrit. (Waldmann) 

Red Cell Life Span Methodology 

DiisopropylfluorophospTiate P^' (DFP^^) was 
evaluated for the measurement of red cell life 
span in the dog and man, and found to be very 
satisfactory. A new method for producing a 
cohort of labelled cells utilizing both cold DFP 
and radioactive DFP ^^ was developed. This 
method involves the administration of a large 
(blocking) dose of cold DFP followed in several 
days by a dose of labeled DFP^^ The DFP^^ 
labeling only those cells which were synthesized in 
the time interval following the administration of 
the cold DFP. In the dog, the red cells produced 
in the first six days in response to an acute 
hemorrhage (15% blood volume) have a shortened 
survival, but the cells produced by the ninth day 
have a normal survival. (Berlin) 

Clinical Studies of ErytTiropoiesis 

In man, studies of erythropoiesis including 
measurement of total red cell volume, rate of pro- 
duction of red cells and red cell life span were 
carried out in patients with multiple myeloma, 
macroglobulinemia, Hodgkin's disease and chronic 
myelocytic leukemia. In multiple myeloma, the 
predominant pattern of ei"ythropoiesis was that of 
erythropoietic insufficiency with a moderate 
shortening of red cell survival. The red cell sur- 
vival was finite but short. Variations in these 
patterns were fomid depending upon the stages 
of disease. Androgen therapy in large does pro- 
duced an increase in the rate of production of red 
cells and an increase in the total red cell volume 
in four of five patients studied. In chronic myelo- 
cytic leukemia a spectrum of patterns of erythro- 
poiesis emerged. This varied from a normal total 
red cell volume, normal rate of production of red 



cells, and normal red cell life span through in- 
creased to decreased rate of production of red 
cells; a finite shortening of red cell life span in 
the order of 75 to 80 days, and later markedly de- 
creased erythrocyte survival in the acute myelo- 
blastic stage with a virtual absence of red cell 
synthesis. 

In macroglobulinemia, several distinct pat- 
terns were found. At one extreme, two patients 
with erythroid aplasia were observed, and at the 
other extreme marked shortening of the red cell 
life span. In two patients, shortening of red cell 
life span could be related to clmical activity of the 
disease and the concentration of serum macro- 
globulins. A preliminary review of studies in 
nine patients with Hodgkin's disease indicates that 
shortening of red cell life span is the predominant 
abnormality except in the late stage where there 
is bone marrow failure. 



LABORATORY OF BIOCHEMISTRY 

During the past year the Laboratory of Bio- 
chemistry was divided into five sections: 

Cytochemistry, Head, Dr. Dean Burk 
Nucleic Acids, Head, Dr. Walter C. Schneider 
Nutrition and Carcinogenesis, Head, Dr. 

Harold P. Morris 
Protem Chemistry, Head, Dr. Elbert A. 

Peterson 
Tumor-Host Relations, Head, Dr. Robert E. 

Greenfield 



Cytochemistry Section 

Uridine and certain 5-fluorinated derivatives 
constitute useful tools for the study of the role of 
inorganic phosphoi'ous in regulating cell metabo- 
lism. The glucose requirements for anti-tumor 
action of these substances were examined to de- 
termine the possible chemotherapeutic applica- 
tion. Triphosphopyridme nucleotide was shown 
for the first time to effect glycolysis by both living 
cancer cells and subcellular fractions thereof. 

Using glycolj'tic assay procedures a naturally 
occurring cytocidal complement-dependent anti- 
body system has been shown to be lacking in the 
blood of germ-free pigs but was present in natu- 



16 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



rally contaminated animals maintained on the 
same diet. With cells of the reticuloendothelial 
system the role of endotoxins in increasing non- 
specific (phagocytic) and specific (antibody) 
resistance to infection was investigated. It has 
been shown that endotoxins of gram negative bac- 
teria exert profound effects on the metabolism of 
mammalian cells by their insulin-like effect on the 
hexokinase reaction. Endotoxin and insulin ap- 
peared to act at the level of mitochondrial bound 
hexokinase, both stimulating mitochondrial gly- 
coUysis. Mitochondrial suspensions prepared 
from mouse melanomas, ascites tumors and brain 
displayed liighly significant glycolytic activities 
which were sensitive to insulin : anti- insulin hor- 
monal regulation. The degree of sensitivity to 
hormonal regulation corresponded approximately 
to that obtaining in white cells of the tissue from 
which the mitochondria were obtained. Data 
obtained from a series of rat hepatomas indicated 
that in these tumors, as in malignancies of other 
tissues, with the development or progression of 
the malignant state there was an increase in capac- 
ity to glycolyze which was associated with decreas- 
ing sensitivity to restraint by anti-insulin 
hormones. 

Effects of vincaleukoblastine (VLB) on glycol- 
ysis and respiration of ascites tumor cells in vitro 
have been demonstrated with several lines of the 
L1210 mouse leukemia and in the Ehrlich carci- 
noma. Protection against the effects of VLB on 
aerobic glycolysis of thioguanine resistant cells by 
monosodiumglutamate and L-arginine hydro- 
chloride were observed. In line with reports of 
glutamic acid and arginine protection against 
VLB with leukemic cells in tissue cultui'e and 
Ehrlich ascites cells in vivo, it was concluded that 
this action of VLB involved gross metabolic pa- 
rameters, glycolysis and respiration, basic to the 
production of cell energy. 

Procedures employing trypsin and DNAase 
were developed for preparing suspensions of large 
number of single viable and metabolically intact 
cells from a variety of solid tumor sources which 
made possible the measurements of metabolic or 
other parameters in the absence of contamination 
with host or necrotic tissues. Normal and myelo- 
blastic leukocytes were found to contain myelo- 
peroxidase and a cytochrome b-like pigment but 
to be relatively low in cytochrome c or oxidase. 



The lethal effects of intraperitoneally injected 
thermophilic chlorella cells into mice seemed to re- 
sult from the induced carbohydrate depletion of 
the animal as well as the possible toxins liberated 
by the plants. 

Nucleic Acids Section 

Studies of the synthesis and metabolism of nu- 
cleic acids have centered around precursors of 
deoxyribonucleic acid (DNA). Basic to much of 
this work has been a microbiological assay which 
used L-acidophilus Ii-26 for the determination of 
deoxyribosidic compounds. Ribonucleotides were 
found to be potent inhibitors of the assay when 
deoxyribonucleotides were being measured but 
this inhibition could be avoided by hydrolyzing 
the nucleotides to the non-inhibitory nucleosides 
with snake venom. This was the basis for an im- 
proved assay procedure. 

The investigation of the occurrence of large 
quantities of deoxycytidine diphosphate clilorine 
in the Novikoff hepatoma was continued. Enzy- 
matic studies of the synthesis of this compound 
and of its utilization for the formation of lecithin 
showed that the synthesis of deoxycytidine di- 
phosphate choline was about the same in the tumor 
as in normal liver but that the ability to form 
lecithin was only one third as great in the hepa- 
toma as in normal liver. These results indicated 
that the high levels of deoxycytidine diphosphate 
choline in the Novikoff hepatoma were due to the 
decreased utilization of this compound for lecithin 
formation, perhaps indicating a mechanism by 
which the tumor can provide a larger pool of pre- 
cursors for DNA synthesis. The intracellular 
localization of these enzymatic activities was also 
studied. Preliminary findings indicated that the 
synthesis of lecithin occurred in the microsome 
fraction while the formation of deoxycytidine di- 
phosphate choline was catalyzed by the soluble 
fraction of normal rat liver. 

Studies on the occurrence of deoxycytidine, 
deoxyuridine and 5-methyl deoxycytidine in the 
urine of rats were continued. Improved pro- 
cedures for the determination of the four 
deoxynucleosides without prior isolation and 
purification were developed which made use of 
an E. coli mutant 70 V-464, L-acidophiliis E-26, 
and chromatography on XE-64 resins. On the 



NATIONAL CANCER INSTITUTE 



17 



same experimental regime females excreted one 
fifth the amount of deoxyribosyl compomids of 
males, an miexplained observation. In both sexes, 
excretion in the urme was elevated on the day fol- 
lowmg partial hepatectomy. The increase was 
not seen in pair fed controls or after 20% partial 
hepatectomy. The data obtained agreed with 
other work indicating that the synthesis of DNA 
during liver regeneration was ahnost complete 
withm 24 houi-s. 

Four seperate kinase systems responsible for the 
phosphorylation of thymidine monophospliate 
(TMP), deoxycytidine monophosphate, deoxy- 
guanosine monophosphate (dGMP) and de- 
oxyadenosine monophosphate, respectively, and 
required for total DNA synthesis were studied 
simultaneously in normal mouse liver cells and in 
mouse ascites hepatoma cells. The limiting kinase 
activity m both hepatoma and liver cells was 
found to be TMP kmase. However, when j)hos- 
phorylation was studied, only 10-15% of the TIMP 
was converted to the di- and triphosphate by 
normal cells while sixty percent conversion was 
obtained with the ascites hepatoma cells. A simi- 
lar but more exaggerated situation was observed 
with dGMP in that nearly complete conversion 
to the di- and triphosphates occurred in the hepa- 
toma cells. The results suggested that liver cells 
possessed a mechanism for controlling di- and tri- 
phosphate synthesis which was absent from the 
hepatoma cells. The hepatoma cells also ap- 
peared to have fewer degradative processes than 
normal liver cells. 

As part of a continuing study of the structure 
and sequences of nucleic acids, chromatograpliic 
teclmiques were used to effect separation of 
nucleotides resulting from enzymatic degradation 
of nucleic acids. Members of a homologous series 
isolated from digests of synthetic polyadenylic 
acid have been obtained in sufficient purity and 
yield to permit a study of the relationship between 
nucleotide chain length and secondary structure. 
The hypochromic effect appeared with the dimer 
and remained constant with increasing chain 
length after the trimer. At a chain length of 7 
and above, evidence of polymer-like secondary 
structure has been obtained from spectral shifts 
and melting curves. Sephadex and ion exchange 
Sephadex have potential use for the fractionation 
of oligonucleotides. Variation in the concentra- 



tion of salt and eluting medium, introduced an 
additional parameter which has produced advan- 
tageous subfractionation. 

Partial and complete enzymatic digestion 
coupled with chromatographic teclmiques have 
been used to characterize eiizjunatic activity, to 
follow the purification of specific enzymes and to 
compare ENA samples derived from different 
sources. Examination in this manner of three 
different strains of tobacco mosaic virus (TilV) 
have clearly distinguished the ENA of the HE 
strain from that of the TMV- and M-strain in 
some of their nucleotide sequences. Similar dif- 
ferences have been reported for the protein com- 
ponent of the TMV virus obtained from these 
strains. 

Since progress in obtaining the nucleotide 
sequences of a nucleic acid depends on specific 
cleavage along the chain, the search for nucleases 
with hydrolytic activities directed toward a spe- 
cific nucleotide linkage was continued. A method 
for the preparation of ribonuclease Tl (specific 
for guanylic linkages) has been developed. 
Quantitative isolation and characterization of the 
mono-, di-, tri-, and tetranucleotides found in 
partial and complete ENA digests produced by 
the action of this enzyme indicated a strict speci- 
ficity for guanylic linkages, since only 3'-guanylic- 
terminated oligonucleotides were found. Eibo- 
nuclease To, reportedly exhibiting specificity for 
adenylic linkages, was purified. Eesults indi- 
cated a preferential splitting of adenylic linkages 
but did not support a strict specificity. 

Purification and characterization of the acid and 
alkalme ribonuclease activities of spleen uncovered 
at least two enzymes which fractionated very 
closely with but could be removed from the ribo- 
nuclease activities, an acid phosphatase which 
readily dephosphoiylated adenylic acid, and a 
phosphodiesterase without ribonuclease activity, 
which hydrolyzes cyclic adenylic acid to yield 2'- 
adenylic acid. Although at complete digestion 
both ENases had hydrolyzed yeast ENA through 
the cyclic mononucleotides to the 3'-nucleotides, 
distmct preferential hydrolysis was observed at 
intermediate stages, the acid ENase digest con- 
taining little or no cytidylic acid, whereas the 
alkaline ENase digests showed veiy small amounts 
of adenylic acid. 



18 



AJSnsrUAL REVIEW OF INTRAMURAL RESEARCH 



The ribonuclease activities of homogenates and 
active nuclease preparations from normal rat 
liver, Novikoff hepatoma and hepatoma 3683 
(Morris) were examined over a pH range of 4.8 
to 9.0. Tlie addition of the sulfhydryl reagent, 
parachloromercuribenzoate (CMB) increased the 
alkaline ribonuclease activity of the homogenates 
2-3 fold by releasing the enzyme from a previously 
recognized inhibitor. Hepatoma 3683 showed 
less KNase activity than normal liver or Novikoff 
hepatoma both before and after the addition of 
CMB. The acid ribonuclease activities, however, 
were inhibited by CMB and this inhibition could 
be reversed by excess cysteine or glutathione. 
Further evidence in support of the sulfhydryl 
dependence of the acid ENase was found in a 
definite correlation between the rate of inhibition 
of activity and the rate of formation of the mer- 
captide as titrated spectrophotometrically. 

Nutrition and Carcinogenesis Section 

Transplantable tumors whose enzymatic com- 
plement showed a minimum deviation from that 
of the normal tissue have been induced in a num- 
ber of ways. The prototype of the minimal devia- 
tion tumor, the transplantable malignant liver 
neoplasm (Morris 5123), was studied in 25 differ- 
ent laboratories throughout the country. Through 
the cooperative effort of this group of interested 
biochemists a large number of unusual enzymatic 
characteristics have been uncovered in this tumor 
as well as in the newer neoplasms which have been 
developed. The Morris 5123 tumor was devel- 
oped in an inbred strain of Buffalo rats available 
only at NIH and a large operation has been re- 
quired to make these tumors available to other 
investigators. Four sublines of this tumor have 
been established and it has already appeared that 
differences in their enzymatic complements have 
developed. Studies on the "minimum deviation" 
tumors so far completed suggested that extreme 
caution must be used in the interpretation that 
there is a "convergence of enzyme patterns to a 
single type" in the development of liver cancer. 
Chemical induction of tumors in inbred strains of 
rats was especially important since it permitted 
the development and transplantation of slow 
growing tumors which otherwise might not have 



survived in non-inbred rats and would have been 
permanently lost. 

Contrary to the general belief that only negli- 
gible amounts of catalase existed in tumor tissue, 
the newer liver neoplasms that have been devel- 
oped had high levels of catalase. A transplant- 
able hepatoma mduced by feeding a simple aro- 
matic amine, 2,4,6-trimethylaniline grew very 
slowly and exhibited a high catalase activity. 
The recently developed Eeuber tumor induced by 
ingestion of 2-fluorenyldiacetamide which grew 
much more slowly than two neoplasms induced 
by this chemical in this laboratory many years 
ago, also possessed catalase activity. The more 
recently developed and slow-growing neoplasms 
in general seemed to have a high catalase content 
whereas catalase activity was absent from almost 
all transplantable liver tumors previously studied. 

The metabolism in various susceptible and 
resistant species of the carcinogen N'-2-fluorenyl- 
acetamide (2-FAA) labelled with carbon-14 has 
been investigated using biochemical, pharmaco- 
logical, histochemical and tracer techniques. 
Physical-chemical studies on the carcinogenic 
metabolites have been performed and the binding 
of the carcinogen to proteins of the liver and their 
separation by chromotography has been initiated 
(J. H. Weisburger, E. K. Weisburger and H. P. 
Morris). Substitution studies on the hydrogens 
of the omega carbon atom of 2-FAA series indi- 
cated that substitution of the hydrogens by the 
halogen fluorine, in the 2 and 7 position of the 
fluorene molecule increased carcinogenic activity. 
In fact, two transplantable tumors from the same 
liver have been induced by ingestion of this chem- 
ical. These neoplasms had strikingly different 
growth rates and the preliminaiy indications were 
that the more rapidly growing tumor had a carbo- 
hydrate enzyme pattern similar to that of the 
Novikoff hepatoma. 

Previous studies of the mechanisms of carci- 
nogenesis indicated that hydroxylation and con- 
jugation were metabolic i^rocesses concerned M'ith 
the detoxification and excretion of the adminis- 
tered carcinogen. The blocking of the usual sites 
of hydroxylation of simple aromatic amines 
yielded information which suggested that inter- 
ference with this process increased cai'cinogenesis, 
in some instances at least, and induced transplant- 



NATIONAL CANCER INSTITUTE 



19 



able tumors having valuable metabolic 
characteristics. 

A compound related to 2-FAA, the 2,7 deriva- 
tive, ]Sr,]Sr,2-7, tluorenyl-bis-acetamide, (2,7-FAA), 
has been shown to be a very active carcinogen 
when compared with 2-FAA. The results of one 
series of experiments with rats have been pub- 
lished as J.N.C.I. monograph No. 5. Some of the 
most important findings included the induction of 
tumors of the small intestines and of the 
glandular portion of the stomach, salivai-y gland 
tumors, and neurogenic tumors. This appeared 
to be the first instance of induction of gastric 
carcinoma by an orally adminstered carcinogen. 
Extensive atrophy of many organs was found in 
those rats that ingested 2,7-FAA. 

The rate of tiunor induction and growth on ade- 
quate diets of natural foodstuffs and on diets of 
similar composition made from pure or chemically 
defined ingredients was compared at isocaloric 
intakes. The results indicated that the intake 
of the simulated diet was definitely poorer than 
that of the natural diet with resultant poorer 
growth. Wlien, however, equal but limited 
amounts of each diet were fed daily, such that 
all were consimied, the growth rates were equiva- 
lent. Partial underfeeding was employed during 
the carcinogenic period. 

Fischer strain rats receiving 2-FAA plus added 
DL-tryptophan and propylthiouracil developed 
liver, ear duct and bladder tumors. Bladder 
tumors could also be induced in Fischer strain 
rats by 2-FAA in the absence of added ti-ypto- 
phan. Previous studies indicated that rats in- 
gesting the 2-FAA carcinogen had an increased 
requirement for pyridoine and that low levels of 
dietary pyridoxine resulted in an increased induc- 
tion period although the incidence of liver tumors 
was essentially imchanged. Continuation of these 
studies with diets containmg 2-FAA and high in 
pyridoxine permitted the development of mam- 
mary tumors earlier than those on the low pyri- 
doxine diet without significantly changing the 
incidence of liver tumors. The difference in liver 
tumor incidence between male and female rats on 
tliis regimen disappeared if the mammary tumors 
of females were removed surgically although 
studies during the precancerous period mdicated 
that histological changes in the liver occurred 
sooner and more extensively in male rats than 

643351 — 62 3 



female rats. Testosterone was able to accelerate 
the early liver histological changes of castrated 
male rats over that of intact males. 

Biochemical studies of experimental cancer have 
shown that the metabolism of 2-FAA by the rhesus 
monkey resembled that of the guinea pig and not 
of the rat. The similiarity of the metabolic pat- 
tern to that of the guinea pig suggested that the 
rhesus monkey was relatively resistant to the car- 
cinogenic action of 2-FAA. No tumors were 
found after six months of contmuous feeding. 

Examination of the glutamic-oxalic trans- 
aminase activity (GOT) of the JNIorris hepatoma 
5123 showed an activity several-fold greater in 
serum and liver than that of normal animals. 
The Reuber tumor induced by N-2-fluorenyldi- 
acetamide has a low GOT activity. Passage of 
the Morris hepatoma for several transfers in 
tissue culture and then reinoculation into rats has 
resulted in approximately a two-fold increase in 
growth rate. The GOT activity, however, seemed 
to be at the same level as in the slower growing 
tumor lines not passed through tissue culture. 
The Dunninghepatoma however, which grew as 
rapidly as the tissue culture grown 5123 tumor, 
showed a low GOT activity. Examination of the 
subcellular distribution of GOT activity in normal 
liver and in hepatoma indicated a different distri- 
bution of activity in neoplastic tissue, with more 
activity in the supernatant fraction of the 
hepatoma. 

The enzyme responsible for the hydrolysis of 
D or L peptides seemed to be localized for the most 
part in the microsome fraction of rat kidney. 
The sum of the various fractions, however, far 
exceeded that of the homogenate. This discrep- 
ancy was found to be due to the presence of a 
powerful mhibitor or series of inhibitors in the 
soluble fraction of the homogenate. The inhibi- 
tor system has been shown to be sensitive to the 
sulfhydryl reagents or simple aerobic incubation. 
Cysteine was a powerful reversible inliibitor. 
Cobalt, and to a lesser extent other cations, over- 
came the action of the inhibitor and further acti- 
vated the hydrolysis of glycyl-D-amino acids. 

Protein Chemistry Section 

Since the effectiveness of chromatograpliy in its 
various applications to macromolecules depends 



20 



ANNUAL REVIEW OF rNTKAMXJRAL RESEARCH 



upon appropriate adjustment to the properties of 
the substances under study, various types of ad- 
sorbents have been developed for those macro- 
molecules which resist resolution by the existing 
cellulose exchange adsorbents. A new exchanger, 
ECG-cellulose, was made by attaching glycine 
molecules to cellulose through the amino groups 
and it possessed both positive and negative 
charges. Because of their proximity to each 
other, these charges were less effective in binding 
polyelectrolytes and permitted desorption with 
low salt concentrations in a more physiologic pH 
range. Polyglucosamine, obtained by controlled 
hydrolysis of deacetylated chitin, was investigated 
as another source of charged groups for attach- 
ment to cellulose. Appropriately sized glucosa- 
mine polymers, separated on cation exchange 
cellulose was used to prepare a series of adsorbents 
having widely separated clusters of uniform 
charges. These adsorbents should avoid the ex- 
cessively tight binding encountered with some 
large molecular weight nucleic acids and 
nucleoprotems. 

Polyamino acids are being used as model pro- 
teins to investigate the effect of increasing size on 
binding to anionic and cationic adsorbents. 
Homologous polyamino acids ranging in size from 
n-2, to n-15-20 have been resolved from polymeric 
mixtures of lysine, glutamic acid, aspartic acid, 
and mixed polymers of tyrosine and glutamic acid. 
These studies have demonstrated that chromato- 
graphic analysis of polymer mixtures can be used 
to characterize the molecular weight distribution 
in the mixture and thus to study the kinetics of 
polymerization and of acid or enzymatic hydroly- 
sis. Large scale isolation of individual members 
of a given series will permit use of a given chain 
length or chain sequence in physical-chemical and 
biological studies. 

Examination of serum proteins has continued 
with emphasis on the chromatographic purifica- 
tion of factor VIII (antihemophilic factor). 
Starting with Blomback FIO fraction, high puri- 
fication has been achieved with recoveries of 85- 
100%. Factor "VIII could also be adsorbed on 
DEAE-cellulose directly from diluted fresh 
plasma and recovered in high yield by elution with 
sodium chloride. Factor V and prothrombin, 
emerging before factor VIII, were also recovered 
in good yield. These factors are required for 



research and clinical applications, and their ex- 
traction directly from diluted plasma should in- 
crease their availability for experimental use. 

Close chromatographic examination of bovine 
plasma albumm provided evidence for various 
types of heterogeneity of the albumin molecule. 
Besides mercaptalbumin (1 mole SH per mole), 
the major component of sermn, three other mono- 
meric albumin components have been observed 
which can be differentiated by their SH content. 
In addition, dimers and higher polymers have 
been detected and concentrated. Two different 
reactions are involved in the ethanol-induced di- 
merization of albumin, of which only one requires 
the participation of free-SH gi'oups. Prelimi- 
nary data with hiunan plasma albumin suggest a 
similar degree of heterogeneity. 

Of the fifteen /^-hydroxy amino acid derivatives 
tested for inhibition in three microbiological 
screening systems, the N-chloroacetyl derivative 
of hydroxymethylserine, a-methylserine, and hy- 
droxyleucine produced growth inhibition. The 
behavior of the three hydroxyamino acid deriva- 
tives was quite different from that of the amino 
acid, in which the zwitter-ion characteristics were 
present. The size and charge of the acyl blocking 
group plays some role in this inhibitory action. 

The cytochemical organization of normal and 
malignant cells was studied primarily in liver 
cells and in plasma cell neoplasms. Microsome 
fractions prepared from the latter elicited good 
antibodies which were usually highly specific 
against the corresponding circulating myeloma 
protein. Particular attention has been devoted 
to those neoplasms which produced high concen- 
trations of urinary proteins in tumor-bearing mice, 
analogous to the Bence-Jones protein in multiple 
myeloma. The Potter plasma cell neoplasm 
(EPC-20) produced urinary protein primarily 
with a sedimentation rate of 2. 9S but with some 
4. 9S material present. The larger component 
could be converted to the smaller material by 
mercaptoethanol treatment. A strong antibody 
to the native urinary protein has been produced 
in rabbits by injecting rat microsomes. It failed 
to react with the normal serum components of 
mice or with any other mouse myeloma protein 
examined. The antigen was located in the endo- 
plasmic reticulum ; none appeared in the ribonu- 
cleoprotein particles. 



NATIONAL CANCER INSTITUTE 



21 



Beta-glucuronidase activity was used as a bio- 
chemical marker for several strains of C3H mouse 
cell tissue cultures in the study of spontaneous 
or directed transformation of these cells in vitro. 
Four new C3H liver cultures which had been 
mitiated have produced one culture line with a 
very high activity, and three other lines with a 
low activity of /?-glucuronidase. 

Fractionation of the ribonucleoprotein particles 
of rat liver by chromatogTaphic and centrifugal 
techniques has resulted in two ribonucleoprotein 
peaks differing significantly in sedimentation co- 
efficients and the ratio of protein to nucleic acid. 
Stepwise elution procedures producing extremely 
sharp ribonucleoprotein peaks made feasible the 
rapid isolation of ribosomes on a large scale. In 
the presence of liigh concentrations of magnesium, 
tight binding to the adsorbent occurred, appar- 
ently as a result of aggregation of the ribosomes. 
In the absence of magnesium the ribosomes dis- 
sociated into large and small subunits. Attempts 
to isolate the large subunit were frustrated by the 
tendency to reassociate under the conditions em- 
ployed. The possibility of chromatogTaphic isola- 
tion and fractionation of mitochondria was inves- 
tigated since it had recently become possible to 
adsorb mitochondria on ECTHAJM-cellulose and 
remove them with a nonionic detergent. 

Tumor-Host Relations 

A method for the isolation of single cell suspen- 
sions of various tissues of the rat was described in 
last year's report and involved intravenous injec- 
tion with tiypsin and dispersion of the cells with 
solution of 10% sucrose and 20% polyvinylpyr- 
rolidone (PVP). Under these conditions viable 
cells were isolated in high yield, relatively free of 
other cell types, and did not release proteins or 
enzymes into the solution. More recently, the 
samples of PVP were highly toxic and caused cell 
disruption. The molecular weight distribution of 
PVP of around 25,000 was fomid to be satisfac- 
tory for the separation of cells but those with the 
molecular weight of less than 10,000 or around 
60,000 were not. The toxic component in PVP 
could be removed by Dowex-1 or by boiling in 
hydrogen peroxide solution. Kemoval of the toxic 
substance again enabled cell preparation to be pre- 
pared which exhibited constant respiratory rates 



over periods of 3-4 hours. Isolated hepatic cells 
have shown respiratory rates of 30 to 60 micro- 
liters per hour and were still capable of anaero- 
bically reducing tetrazolium salts intracellularly 
to f ormozan after four hours incubation. Hepatic 
cells maintained at 37° under culture conditions 
showed a "tree-like" organization attached to the 
bottom of the culture vessel. This organization 
did not occur at lower temperatures or at unphys- 
iologic pH levels. 

Tumor cells isolated from lymphosarcoma 
E2788 and hepatoma 3683 grew well on reinocula- 
tion into compatible hosts. Metastases to the 
lungs were noted with intraperitoneally injected 
cell suspensions but not with imdispersed cells. 

Close correlation between the activity of the 
enzyme lactic acid dehydrogenase (LDH) in the 
plasma and the growth of a variety of different 
types of mouse neoplasms has been fovmd by Eiley 
in animals bearing tumors which have been trans- 
planted for many generations. However, animals 
with spontaneous tumors or transplanted tumors 
of more recent origin were not associated with 
markedly elevated plasma LDH activity. The 
plasma LDH was lost when the Moloney leukemia 
virus was passed through rats and then back into 
mice although the oncogenic properties were main- 
tained. Diverse findings suggested that the trans- 
missable agent was probably a virus of mouse 
origin being carried along by a number of trans- 
planted tumors. 

A study of the in Advo environment of the cells 
has been continued utilizing "tissue isolated tu- 
mors" (P. M. GuUino) with an examination of 
the in vivo production of connective tissue prob- 
lems by rat and mice tumors. It has been observed 
that the ratio between total proteins and those of 
connective tissue was constant for each tumor over 
many transplant generations. Epithelial cells of 
Novikoff hepatoma grown m millipore chambers 
placed into the peritoneal cavity of the host were 
imable to form collagen but did produce an 
amount of connective tissue proteins characteristic 
of Novikoff hepatoma when grown "tissue iso- 
lated." Thus it was demonstrated that the con- 
nective tissue proteins must have been formed by 
the host. Transplantation of the same cell popu- 
lation into different strains of rats led to tiunors 
with an amount of comiective tissue protein which 
varied from one strain to another, whereas that 



22 



ANNUAL EEVIEW OF INTRAMURAL RESEARCH 



Avithin a host strain was constant. It Avas possible 
to separate two lines of tumor producing cells 
from one hepatoma which diJffered two-fold in the 
connective tissue protein content. The results 
were interpreted as indicating that hepatoma cells 
"stimulated" production of connective tissue pro- 
tein by the host and that the amoimt formed de- 
pended on the cell type of the hepatoma. 

The interaction between tumor cells and host 
cells and the potentialities for growth differentia- 
tion of the free cells of the reticulo-endothelial 
system (lymphocytes, macrophages, polymorphs 
and mast cells) were examined in diffusion cham- 
bers implanted intraperitoneally into the rat. 
Pretreatment of the chambers by placing them 
empty into mice before adding the lymphocytes 
provided better growth and survival than if the 
cells were put directly into freshly prepared 
chambers. Lymphocytes exhibited better growth 
and morphological integrity if they were grown 
in chambers together with connective tissue. 
Thoracic duct lymphocytes have also been studied 
with these techniques. 

Tumor cells from the rat and mouse have been 
separated from host cells by serial transplantation 
of the tumor cells from chamber to chanaber. The 
connective tissue cells remained fixed to the milli- 
pore filter and the tumor cells could be scraped off 
and transplanted. Connective tissue was diluted 
out after three such transplantations. Novikoff 
hepatoma cells (low connective tissue formers) 
prepared in single cell suspensions by the trypsin- 
PVP-sucrose techniques were cleanly separated 
from all stroma cells by serial transplantation in 
the chambers. However, hepatoma 5123 and an 
ethionine-induced hepatoma, both of which pro- 
duced dramatic comiective tissue response, were 
much more resistant to the separation of connec- 
tive tissue from tumor cells because of the limited 
growth potential of the tumor and the very inti- 
mate association between tumor islets and the 
comiective tissue. 

Normal C3H mouse connective tissue could be 
grown for longer periods of time in diffusion 
chambers implanted into C3H mice than in tissue 
culture. Thus fibroblasts which were removed 
from the chambers after 12 months growth and 
cultivated in vitro, became malignant after 3 
months (or a total of 15 months of isolation from 
the mouse). Wliereas the fibroblasts that were 



allowed to remain in the chambers were still nor- 
mal after 18 months. After 23 months, however, 
the fibroblasts grown only in the chambers also 
produced tiunors upon injection into C3H mice. 
It was of interest that transplantation of the tissue 
from chamber to chamber was not an inducing 
factor in this transformation. Tissue kept in the 
same chamber for 378 days produced tmnors as 
rapidly and with the same incidence as tissues that 
were transplanted 11 times during the same 
interval. 

Studies on the kinetics of catalase synthesis and 
destruction have indicated that the lowering of 
liver catalase by tuniors is more nearly duplicated 
by a protein-free diet than by starvation. Hypo- 
physectomized animals showed a level of liver 
catalase similar to that seen in starvation. Hypo- 
physectomized animals bearing thyroid tumors 
exhibited a market decrease in liver catalase indi- 
cating that the effects of hypophysectomy and 
tumor growth were additive and that hypophy- 
sectomy did not block the effect of the tumor on 
liver catalase (M. Kechcigl, Jr.). Utilizing the 
specific uiliibitors, 3-amino-l,2,4-triazole (AT) 
and allyliospropylacetamide (AIA), identical 
rates of catalase destruction in rat liver and kidnej' 
have been demonstrated although the rate of cata- 
lase synthesis in the liver was four times that of 
the kidney. Incorporation of Fe^^ into liver 
catalase was almost completely blocked by AIA. 
Isolation studies however indicated that a small 
fraction of catalase was resistant to AIA. This 
small catalase pool tm'ned over rapidly but so far 
has not been shown to differ from the main catalase 
fraction. 

The finding that certain of the induced rat hepa- 
tomas had elevated levels of catalase activity was 
unexpected as all the tumors previously studied 
had very low activity. Fm-thermore, it had been 
postulated that the tumor itself produced a sub- 
stance "toxohormone", which was capable of lower- 
ing catalase activity of the liver. Studies of the 
catalase in the Morris 5123 hepatoma have indi- 
cated that this tumor had a smaller rate of catalase 
synthesis and a greater rate of catalase destruction 
than the normal liver. 

Catalase may well be a biochemical marlcor with 
which to follow the transformation and differen- 
tiation of tumor tissue. An ethionine induced rat 
hepatoma, arising in OM/N rats, has given rise to 



NATIONAL CANCER INSTITUTE 



23 



two lines of hepatomas : one possessing extremely 
high catalase activity, and the other with low 
catalase activity. The low catalase line gi-ow more 
rapidly than the high catalase line. These differ- 
ences have persisted over ten transplant genera- 
tions. 

Durmg the period of most rapid tumor gTowth 
and despite the onset of anemia, rats bearing 
lymphosarcoma E2788 gained appreciably more 
weight than normal controls with almost identical 
food consumption. Eats bearing hepatoma 3683, 
on the other hand, consumed less food and gxew at 
equal or slower rates than the controls. Both types 
of tmnors, however, exerted similar effects on the 
host with regards to the development of anemia, 
elevation of plasma aldolase and depression of 
liver catalase, etc. Carcass and tumor analysis 
showed that the lymphosarcoma bearing animals 
had sufficient water retention to explain their in- 
creased weight gain but an equally high degree of 
hydration was observed with the hepatoma bear- 
ing rats whose body weight gains were lower than 
those of the normal controls. Onset of anorexia 
and negative nitrogen balance was not prevented 
by an increase in the sodium chloride intake nor 
was there any apparent gain in weight, although 
the fluid consumption of both tumor groups had 
increased. In fact, a decreased survival time was 
observed among tumor bearing animals ingesting 
saline. In free choice experiments, neither tumor 
bearing rats nor control animals showed a prefer- 
ence for fluid containing sodium chloride. 



LABORATORY OF BIOLOGY 

The research program of the Laboratory of 
Biology presents a rather broad biological ap- 
proach to the problem of cancer. Primary in- 
terest is in the etiology of cancer with patho- 
genesis and therapy through the biological 
approach also receiving some consideration. The 
host is given equal consideration to that of the 
etiologic agent and the developing neoplasm. 

In a summary it is impossible to list all findings 
given m the individual reports, but the reviewer 
has attempted to select those which to hun ap- 
pear to illustrate best the scope of the program 
of the laboratory. Findings are grouped under 
six headings (carcinogenesis, immimology, virol- 



ogy, genetics, drug resistance, and pathogenesis) , 
but in most cases the boundary lines are not clear 
and many projects fall within the scope of more 
than one discipline. Furthermore, the work of 
no investigator falls in one category alone. Many 
of the reported findings are the result of collabora- 
tive work between members of the laboratory, with 
scientists in other laboratories of the Institute, 
and with outside uivestigators. 



Carcinogenesis 

Carcinogenesis is considered m its broad biologi- 
cal sense by members of the laboratory. Work 
is directed not only toward further study of the 
effect of extrinsic chemical and physical carcino- 
gens, but also toward further analysis of intrinsic 
factors involved and the nature of the carcinogenic 
process. Carcinogenesis is the central theme of 
the research program of the laboratory. 

In continuation of his woi'k on the role of the 
thymus in leukemogenesis, Dr. Law in collabora- 
tion with Dr. Bradley discovered that in thy- 
mectomized, irradiated CsjBL/Ka mice sub- 
cutaneous, isologous thymic grafts develop into 
lymphomas, whereas those transplanted mider the 
kidney capsule do not. The grafts under the kid- 
ney capsule reconstitute and contmue to grow but 
fail to show any of the morphologic changes char- 
acteristic of thymic tissue transforming to neo- 
plasia. The mechanism of this inhibition is being 
studied further. 

Dr. Potter has contmued the study of induction 
of plasma cell tumors that at last provides a con- 
sistent experimental system for stud5nng the 
pathogenesis of multiple myeloma. Mineral oil 
and mineral oil adjuvants, complete Freund's ad- 
juvant and the adjuvant-staphylococctts mixture 
all injected interperitoneally are effective in induc- 
ing plasma cell timiors on BALB/c mice. Three 
injections of the oil alone results in the induction 
of this neoplasm in 60 percent of the mice. Such 
high frequency permits study of other aspects of 
the induction of this neoplasm including suscepti- 
bilit}' of other strams, influence of genetic factors, 
role of antigen incorporated into tlie oil, and the 
effect of route of administration. This work has 
practical significance in that mineral oil and min- 
eral oil adjuvants have been injected clinically into 
man. However, Drakeol-6VR, the oil approved 



24 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



for this purpose, has thus far resulted in but few 
plasma cell neoplasms in mice. 

Dr. Deringer has added further evidence that 
urethan, once thought to be limited to induction of 
lung tumors, is a multipotential carcinogen. A 
high percentage of hairless strain HE mice painted 
with urethan in ethylene glycol developed epider- 
moid carcinomas compared with only very few of 
those iDainted with ethylene glycol alone. Hepa- 
tomas and hemangioendotheliomas had been 
shown in this laboratory to be induced by urethan, 
and in other laboratories urethan was shown to 
augment the effect of a niunber of other agents in 
inducing leukemia. 

Dr. Heston reported what may be a basic ob- 
servation on the role of the hypophysis on induc- 
tion of neoplasms. (C3H X Y)Fi male mice hy- 
pophysectomized at one month of age were kept 
mider observation until they were 16 months old — 
much longer than any hypophysectomized mice 
had previously, to his knowledge, been observed. 
At that time not one of these males showed any 
neoplasm, although normally 60 percent of them 
would have been expected to have hepatomas. 
This emphasizes the need for further studies of the 
necessity of normal growth processes in car- 
cinogenesis. 

There has been continued interest in the nature 
of the carcinogenic process or the neoplastic 
change in the cell, and one approach has been 
through dose-response studies. Earlier Heston 
and Schneiderman published a straight line i)ul- 
monary tumor response to graded doses of di- 
benzanthracene indicating a single event action 
which, if it were a gene change, should be con- 
sidered as a dominant mutation. Left unex- 
plained, however, was the fact that the extension 
of the line to zero dose fell below the point ob- 
served. Eesponse data for these low doses have 
now been obtained and analyzed in collaboration 
with Mr. Mantel and Miss Gurian. They were 
found to be in accord with the earlier interpi-e- 
tation if it is assumed that each mouse has its own 
threshold for the carcinogen and its own charac- 
teristic sensitivity. The dose-response studies 
were extended to a soluble carcinogen, urethan, 
that also gave a straight Ime response. In addi- 
tion, the study provided an adequate comparison 
of the carcinogenic activity of the two carcinogens. 



urethan, generally considered to be a potent car- 
cinogen, was only one percent as active as the 
dibenzanthracene. 



Immunology 

Work of the laboratory in tliis area generally 
concerns the field presently called "immunogenet- 
ics", which covers an area of overlap between the 
tAvo classical disciplines. 

Dr. Barrett has continued to work with the 
Barrett-Deringer phenomenon of enhancement 
few years ago. The original observation was that 
the tumor grew better in the backcross generation 
after having been transplanted from the parent 
strain of origin into the Fi that differed geneti- 
cally from the parent strain. The effect does not 
hold for all tumors, however, for he reports fail- 
ure of altered transplantability in a recently 
tested C3H tumor. 

Dr. Barrett reports further on the observation 
made in collaboration with Dr. Breyere of toler- 
ance induced in female mice by parity. The ef- 
fect has now been observed in three genetic 
systems with three transplanted tumors, and 
with skin homografts in two combinations. Tol- 
erance can be seen when the materials are the 
same or different at the H-2 locus. In one of 
the tumor-host combinations the tumors in the 
tolerant female displayed an interesting growth 
pattern. After reaching a certain size tlie tumors 
became umbilicated in the center without necrosis 
until they appeared as douglinuts with no tumor 
grossly or microscopically visible at tlie inocula- 
tion site. This observation suggests a "local 
immunity". 

Other observations reported by Barrett are 
that, in contradiction to the idea often held that 
transplantation antigens mature late and are not 
present in newborn mice, he was able to immunize 
suitable mice against the implantation of a tumor 
by prior inoculation of either embryos or placen- 
tas. He points out that this was really a repeat 
of an old and apparently forgotten experiment. 

In collaboration with Miss Uphoff, Dr. Barrett 
has carried out experiments relative to the often 
held concept that transplantation immunity, once 
established, cannot be abrogated by irradiation. 
They used preimmunized mice, lethally irradi- 



NATIONAL CANCER INSTITUTE 



25 



ated, and protected with bone marrow. Their 
results thus far are in contradiction to this gen- 
erally held concept. 

Miss Uphoff has continued her studies of the 
immunogenetic aspects of bone marrow protection 
against x-irradiation. Much of this work is in 
the area of irradiation protection, but certain 
aspects are directly concerned with neoplasia. 
She reports further observations of the liistocom- 
patibility patterns of lymphomas arising in irra- 
diation induced chimeras of AICR mice i-eceiving 
bone marrow from other strains. The lympho- 
mas arising in these mice are primarily of donor 
tissue origin. Some were of host origin but they 
may have arisen from leukemic cells that were 
not destroyed by the radiation. Some appeared 
to be of both donor and host origin. 

Part of Dr. Potter's work on plasma cell tumors 
falls in the field of immunology. Since normal 
plasma cells are the source of humoral antibody 
the question arises as to whether neoplastic 
plasma cells can make antibody. He has trans- 
planted plasma cell tumors from hosts which have 
in their circulation good titres of anti-ovalbumin 
antibody, but to date no precipitating antibody 
production has been adoptively transferred 
through the transplants. 



Virology 

Members of the Laboratory of Biology continue 
to show interest in the viral etiology of cancer. 
The mammary tumor virus that has been studied 
for many years in this laboratory continues to 
demand considerable attention. Dr. Andervont 
now has four sublines of strain Kill from which 
the mammary tumor virus has disappeared or in 
which it has become inactive with the normal 
route of transmission. He is now reporting on 
experiments designed to determine why the virus 
disappears from certain EIII mice but not from 
C3H mice. Some of the results indicate that the 
viruses differ either quantitatively or qualita- 
tively. The EIII virus consistently gives a lower 
tumor incidence in C3H mice than does the C3H 
virus in C3H mice. Other results indicate that 
the mouse strains likewise differ. It appears that 
the C3H virus may also occasionally disappear 
when placed in RIII mice. 



Doctors Deringer and Ileston continue to study 
the development of mammary tiraiors in lines of 
mice deprived of the mammary tumor virus. Dr. 
Deringer has developed her agent-free lines by 
transfer of fertilized ova. She reports that her 
DBA/2eB line without the virus has very few 
tumors in virgin females, but the incidence can 
be increased by breeding and even more increased 
by force-breeding. However, in each case the 
incidence is lower than in the comparable CsHeB 
group. She has also started an RIIIeB strain 
without the virus. Dr. Heston has developed his 
agent-free lines by foster nursing following Cesar- 
ean birth. In a newly established Af line no 
mammary tumors have been observed to date. 
In these agent-free Imes the effect of the hor- 
monal stimulation and of the genes becomes more 
apparent in the etiology of mammary tumors, for 
greater differences in tumor iiicidence caused by 
these factors can be observed. 

Dr. Law in collaboration with Dr. Moloney has 
been studying congenital transfer of the Moloney 
and the Gross leukemia viruses. Transfer is 
through the maternal line with the milk provid- 
ing an efficient means of transfer. They have 
been unable to demonstrate transfer through the 
paternal line. This is in sharp contrast with the 
usual leukemias of the high leukemic strains such 
as AKE and Css, which are transsmitted through 
the male as readily as through the female. A 
reappraisal of the question of whether or not 
viruses can be considered to be involved in the 
etiology of the usual leukemias of these high leu- 
kemic strains is, therefore, indicated. 

Relative to their discovery of histocompatibil- 
ity tolerance induced by parity, Drs. Barrett and 
Breyere in collaboration with Dr. Moloney, have 
found that this tolerance may decrease the latent 
period of the Moloney virus. 

In continuation of their studies of an attenuated 
• strain of the polyoma virus grown m a milk me- 
diimi, Drs. Law and Eabson have observed re- 
adaptation in a serum medium resulting in a re- 
turn of the oncogenic properties, although in the 
milk medium the virus has remained attenuated. 
Some success has been achieved in immunizing 
CsHf/Bi mice against the potent oncogenic strain 
of polyoma virus with the attenuated strain. 

Mr. Melroy has continued to operate liis labora- 
tory for testing the stocks of mice in Biology and 



26 



ANNXTAL REVIEW OF INTEAMXJRAL RESEARCH 



Viral Oncology for polyoma virus. Sixty-one per- 
cent of the tests have been for the Laboratory of 
Biology and 39 percent for Viral Oncology. For- 
tunately most of our colonies of inbred mice are 
free of the virus. 

Dr. Heston and Mr. Vlahakis, m collaborative 
work with Dr. Moloney reported that in seven 
transplanted tmnors carried for years tests for 
oncogenic viruses have for the most part been neg- 
ative. This suggests that despite the fact that the 
Moloney virus was isolated from a well known 
transplantable tumor, oncogenic agents cannot be 
demonstrated by present techniques in most of our 
transplantable tumors. 

Genetics 

Probably the most basic of all research in the 
laboratory is that in genetics. The geneticist is 
concerned with the mformation passed on to the 
ind.ividual at the time of conception and the man- 
ner in which tliis information directs develop- 
mental processes and acts in conjunction with other 
factors to determine whether or not a neoplasm 
will occur. He is interested in the genetic infor- 
mation of the cell characterizing the neoplasm. 
All work of the laboratory is related in some way 
to these problems. 

We must continually be characterizing new 
strains of mice. Dr. Law is characterizing strain 
CsHf/Fg in which he has observed a leukemia in- 
cidence of 80 percent in the females and 50 percent 
in the males. This is principally lymphocytic 
leukemia. Dr. Deringer has further characterized 
strain BL in describing amyloid in all animals of 
the strain at 15 months of age. Dr. Andervont has 
reported the occurrence of tmiaors in his colony of 
wild mice. It is significant that of a total of 225 
mice, 43.5 percent had developed spontaneous 
neoplasms. Lung tumors occurred most fre- 
quently, with reticuliuncell neoplasms nest. Of 
particular interest in respect to the virus etiology 
of cancer was the fact that while the colony is 
known to harbor the mammary tumor virus, only 
6 percent of the breeding females and none of the 
virgin females developed mammary tumors. 

Dr. Law has made a genetic analysis of the dif- 
ference between strain CsyBL/KaB, none of which 



develop thj-mic or parotid neoplasms when in- 
jected with a thjanotropic polyoma virus strain, 
and strain CsHf/Bi, 100 percent of which develop 
such neoplasms when injected with the virus. 'Re- 
sults of hybridization studies suggest that the re- 
sistance to the oncogenic eif ects of tliis virus is due 
to a single dommant gene. 

Doctors Heston and Deringer are approaching 
the problem of the genetics of cancer in a more de- 
finitive mamier by identifying effects of specific 
genes on the occurrence of specific neoplasms. To 
date 8 different genes on G different chromosomes 
have been shown either to increase or inhibit the 
occurrence of lung timiors. The lethal yellow gene 
is most interesting because it can be analyzed in an 
isogenic system. Heston showed that this gene not 
only increased Imig tumors, but also hepatomas 
and mammary tumors. These observations have 
now been confiiined by Deringer, using different 
strains. There is a correlation between the effect 
of the gene on lung tumors and its effect on noz-mal 
growth, and elimination of the effect of the gene on 
normal growth by food restriction, or mjection of 
goldthioglucose likewise eliminated its effect on 
occurrence of the tumors. 

Dr. Potter is laying some ground work for so- 
matic cell genetics in his plasma ceU tiunors. 
Using immunoelectrophoresis and double diffusion 
precipitin methods and straight agar gel electro- 
phoresis he has identified over 20 plasma cell types 
producing different proteins. It may be possible 
to use these different proteins as genetic markers 
in studying gene action in the origin and nature of 
the neoplasm. 

Transformation of leukemic cells by DNA prep- 
arations was reported by Dr. Bradley and Dr. 
Law. The character chosen was 8-azaguanine-re- 
sistance in the P-388 lymphocytic neoplasm. P- 
388 cells incubated m vitro with DNA jjrepared 
from cells with a high level of resistance, regularly 
developed clonal, resistant colonies at a frequency 
nearly 40 times that observed in control cultui-es. 
Further studies by Bradley concerning antagonism 
by DNA extracts from other sources and substitu- 
tion of adenme for DXA extracts points up the 
danger of interj^reting this type of experiment 
without the further investigation of the true na- 
ture of the change. 



NATIONAL CANCER INSTITUTE 



27 



Drug Resistance 

Dr Anderson has continued her work on bio- 
chemical differences in the metabolism of azaser- 
ine-sensitive and -resistant lines of the plasma cell 
neoplasm 70i29. Duazomycia that inliibited 
growth of sensitive cells to 3 percent of that of con- 
trols compared with inhibition by azaserine to 16 
percent of controls, was tested against the azaser- 
ine-resistant tmnor line to look for cross-resist- 
ance to duazomycin. Marked cross-resistance 
was observed. Wliile collaborating with Dr. 
Wallace Brockman in further exploration of the 
biochemical effects of the three inliibitors, azaser- 
ine, duazomycin, and DON, in the sensitive and 
resistant Imes of 70429, she exposed growing tmnor 
cells to radioactive formate in vivo following pre- 
exposure to a single dose of each inlubitor and de- 
termined incorporation of the tracer in the nucleic 
acids. Each of the three compounds inhibited the 
incorporation of the tracer. 

Dr. Anderson has studied uracil anabolism in 
fluorouracil- and fluorouridine-resistant lines of 
mast-cell neoplasm P815, and has observed the 
following pathway : 

12 

uracil=uridine — 5'-ui'idylic acid 

Fluorouracil is apparently anabolized by the same 
path in mammalian cells. Step 2 is catalyzed by 
uridine kinase, and marked decrease in activity of 
this enzyme is associated with resistance. 

In collaborative studies with Drs. Brockman 
and Davidson, Dr. Law has observed deletion of 
enzyme (pyrophosphorylase) activity m anti-pu- 
rine-resistant cell lines. All of these observations 
will probably tie in with somatic cell genetics. 



LABORATORY OF CHEMICAL 
PHARMACOLOGY 

Eesearch in the Laboratory of Chemical Phar- 
macology continues to be progressively re-oriented 
toward what is currently designated as "Molecu- 
lar Biology." The emphasis is on the understand- 
ing of biological problems in terms of the inter- 
actions of the biological systems with chemical 
agents. The latter include compounds of low- 
molecular weight and of unif omi composition, and 
polymers of high -molecular weight. The macro- 



molecules under study are those isolated from liv- 
ing systems, and synthetic ones prepared to serve 
as models of the former. In the context antigens 
and antibodies are considered to be merely macro- 
molecules with specialized biological properties 
in consequence of their physico-chemical charac- 
teristics. 

The several lines of work are not rigidly walled- 
off from each other as unconnected academic en- 
terprises. They rather are interconnected in 
many ways despite the fact that different academic 
disciplmes are the different tools employed. For 
example, increasing attention is being given to 
Viruses: Dr. Mora's group is studying the trans- 
formation in vitro of normal cells to malignant as 
accomplished by viruses which elicit cancer in 
mammalian species ; Dr. Landy's group, in investi- 
gating the question of the presence in cancer 
tissue of antigens not found in the normal tissues, 
has as one working hypothesis that such antigens 
may not be endogenous in the tumor cells but 
rather may reflect the presence of viiiises; and 
Dr. Goldin's group, in laying the basis for chemo- 
therapy of virus-induced tumors in laboratory ani- 
mals, has found it necessary to add an additional 
target, viz., anti-viral chemotherapy. 

Another example is the follow-up on the pio- 
neering work of Dr. Shear with bacterial polysac- 
charides which include hemorrhagic-necrosis in 
tumors of animals and patients. Drs. Oroszlan 
and Mora have made progress in the further sepa- 
ration of the active fraction, from other compon- 
ents, in the tumor-necrotizing polysaccharide ma- 
terial previously px'eparedby Perrault and Shear; 
a 4-fold increase in potency was obtained, and this 
more-concentrated agent makes possible more so- 
phisticated investigation of its chemical properties 
and of the mechanisms of its biological properties. 
Work in the Imrnmiology Section led to the dis- 
covery of a component in normal blood serum 
and in liver cells which inactivates such endotoxic 
polysaccharides; in an extension of these findings 
it was found in the Macromolecular Cliemistry 
Section that the mactivation with an extract from 
liver cells was a conjugation of the anionic poly- 
saccharide with a cationic protein and that this 
inactivation could be reversed with either a more 
strongly acidic or basic jiolymei-. Dr. Landy, in 
collaboration with Dr. Edgar Eibi of the Rocky 
Momitain Laboraton^ of the NIH in Hamilton. 



643351—62- 



28 



ANlSnjAL REVIEW OF INTRAMURAL RESEARCH 



Montana, has made a series of important contribu- 
tions to the currently controversial subject of tlie 
chemical features of endotoxins which are respon- 
sible for their biologic properties. Dr. O'Malley 
has been investigating the mechanisms through 
■which such bacterial polysaccharides damage solid 
tumor tissue; he has been throwing useful new 
light on the responses of the blood vesels in the 
tumors and on the appearance of a hitherto lui- 
known substance in the blood of mice which had 
received a single injection of the active polysac- 
charide from S. marcesceiis. 

The basic science aspects of research in chemo- 
therapy, conducted by Dr. Goldin and his associ- 
ates, have continued to be fruitful both in the 
intramural Biochemical Pharmacology Section 
and in the contract operation with Microbiological 
Associates, Inc. Dr. Goldin dovetails his chemo- 
therapy studies with clinical objectives and works 
in intimate relationship with the clinical investi- 
gators of the NCI. 

The results obtauaed in the three Sections of this 
Laboratory may be sumn:iarized as follows: 

Immunology Section 

Under the leadership of Dr. Landy, various lines 
of work were prosecuted with gratifying success. 
Collaborative investigations were also carried out 
with men in other institutions both here and 
abroad. 

The Tumor Antigen Problem 

In the face of all the negative results over half 
a century, newer work carried out in various parts 
of the world has re-opened this question. Bjork- 
lund (Stockholm) had provided us with a supply 
of anti-serum obtained from the horse after pro- 
longed immunization with i^ooled human car- 
cinoma tissues. This was absorbed here, exhaus- 
tively, with pooled normal human tissues to remove 
antibodies against normal tissues. This treatment 
removed anti-human reactivity without apprecia- 
ble loss of cytotoxicity for Hela cells or of capacity 
to agglutinate erythrocytes coated with Hela cell 
extracts. 

Thereupon, horses of our own were immunized, 
in weekly injections for more than a year, with 
suspensions of fresh Hela cells; for control, im- 
munization was also carried out with pooled sus- 



pensions of normal human cells (kidney, liver, 
and spleen) . Both anti-sera were found toxic for 
Hela cells. While absorption with normal human 
tissue materials removed this potency from the 
antiserum to normal human tissue it left it essen- 
tially unaltered in the anti-Hela serum. These 
antisera, when tested against tanned erythrocytes 
coated with extracts of Hela cells, yielded analog- 
ous results. 

Role of the Antigen in Immonologic Tolerance 

Inunounological tolerance is elicited more 
readily in neonatal anunals than in adults. The 
greater the retention of antigen in neonatal chicks, 
the greater the degree of immonologic tolerance 
(previous work of Dr. Hirata). In the newer 
work S^Mabelled Bovine Serum Albumin (BSA) 
was injected mto neonatal and older chicles. The 
uptake by spleen was greater in the neonatal. He 
found that the antigen, uicorporated in liver, dis- 
appeared at similar rates in the two age groups 
whereas the rate of disappearance from spleen 
differed for the two groups, indicating that the 
material retained in the spleen may be qualita- 
tively different in neonatal chicks than in older 
ones. 

Endotoxin 

Various aspects of endotoxms, and of their 
biological effects, have been investigated. 

It is generally believed that immmiity in the 
classic sense plays no part in tolerance to endo- 
toxin because of the non-specificity of its elicita- 
tion. However, earlier work of Dr. Landy and 
Ms associates showed that natural antibodies to 
Gram-negative bacteria are widely distributed in 
mammalian species, and that administration of 
any endotoxin leads to a prompt elevation of these 
specific antibodies. Hence the previous concept 
of non-specificity was open to question. Intra- 
venous injection in rabbits of colloidal RES 
blockading agents, in doses which promptly 
abolish tolerance, were foimd to cause an equally 
prompt and precipitous fall in circulating anti- 
bodies to endotoxins. Serum from endotoxm- 
tolerant or normal rabbits, after incubation in 
vitro with such "blockading" agents, sliowed a de- 
crease in antibody content. In the past it had 
been believed that these colloidal materials ex- 
erted their effects pi-imarily on phagoc3'tic cells of 



NATIONAL CANCER INSTITUTE 



29 



the EES. These newer findings suggest that they 
may operate through reducing the level of cir- 
culating antibodies in the blood and thus interfere 
with the clearance of endotoxin. This line of 
work provides data for an immunological basis 
for tolerance to endotoxin. 

Work in the Immunology Section on endo- 
genous agents in mammals capable of inactivating 
endotoxin was brought to an appropriate stopping 
point. Of the organs of the perfused rabbit, only 
the liver yielded extracts which consistently dis- 
played high endotoxin-inactivating potency. 
This work, conducted in collaboration with Dr. 
V. Waravdekar of the AFIP, showed that the 
intracellular agent obtained from hver resembled 
in its properties the one (EDC) in blood except 
that it was not demonstrably affected by divalent 
cation. 

In collaboration with Dr. E. Ribi of the 
Montana Laboratory of the NIH, excellent 
progress was made in removing from an endotoxin 
(S. enteriditis) associated materials (lipoidal and 
nitrogenous) which do not contribute to endotoxic 
properties: lipoidal content was brought down 
from about 6 percent to below detectable levels, 
and the nitrogen content from about 6 to approxi- 
mately 0.5 percent. The final product, a high- 
molecular weight polysaccharide, contained only 
small amounts of fatty acids and phosphorus. It 
is noteworthy that the analytical values for this 
highly potent purified endotoxin were virtually 
the same as for the low-molecular weight haptene 
which is devoid of endotoxic properties. Since 
differences in chemical composition were not found 
between endotoxin and haptene which could ac- 
count for the striking difference in their biological 
behavior, it is surmised that physico-chemical fea- 
tures of these complexes may turn out to play the 
critical role. 

It was visualized that host reactivity could be 
dependent on the macromolecular properties of 
endotoxin. A critically large size of the complex 
might be essential, and the loss of potency wliich 
we found to occur within minutes after treatment 
with dilute acetic acid might result from a simple 
depolymerization into particles of the size of 
haptene. 

This line of work on correlation hetiveen struc- 
ture and Viological properties of endotoxin did 
not support a widely-publicized theory that the 



lipid content was I'esponsible for the host re- 
sponses. Wlien differences in chemical composi- 
tion were ruled out, experiments were designed 
to test the physico-chemical hypothesis. 

The endotoxin prej)aration was boiled with 
0.1 N" acetic acid, which rapidly and progressively 
reduced endotoxic activity. Samples of the hy- 
drolytic products were removed at intervals and 
examined in gel diffusion (Ouchterlong) tests and 
in the analytical ultracentrifuge, and also assayed 
for biological potency. The starting endotoxm 
behaved as a single entity in the ultracentrifuge. 
As hydrolysis progresses, a second, much slower 
moving boundary appeared and, as the fast mov- 
ing endotoxin boundary decreased, the area mider 
the slow moving boundary, subsequently shown to 
be haptene, increased correspondingly. The gel 
diffusion tests likewise provided evidence of a 
progressive increase in the concentration of hap- 
tene during the exposure of endotoxin to acid. 

The findings obtained with these parameters 
were strikmgly consistent and revealed that the 
rate at which host reactive properties were abol- 
ished paralleled the rate of dissociation of the 
complex into haptenic units, particles whose size 
was of the order of 1/100 that of the original 
endotoxin. 

Potent endotoxin can exist in aggregates rang- 
ing all the way from 190 Svedberg units down to 
about 10 S ; within this spectrum there has not yet 
been established a correlation between size and 
potency. Likewise, the size of the aggregate in 
the original endotoxin does not appear to affect 
the rate of destruction of potency nor the dissoci- 
ation of endotoxin into uniform particles of the 
size of haptene. These findings give no indication 
of the formation of particles of intermediate size 
during the reduction of endotoxin to haptene. 
Eather, it would seem that cleavage of endotoxin 
by acid yields haptene directly, i.e., dissociation of 
an aggregate into its primary component particles. 
These findings suggest that the major require- 
ment for endotoxin to elicit the spectrum of host 
reactions may well be a macromolecular complex 
of critical size. 

Further woi-k, in collaboration with Dr. Mark 
Woods, was carried out on the effects of endotoxin 
on the metaholhrn of normal majnmalian cells. 
Tlie earlier findings on tumor cells were extended 
to include an array of normal mammalian cells. 



30 



ANNXJAL REVIEW OF nSTTRAMXJRAL RESEARCH 



Similar stimulation of glycolytic activity by endo- 
toxin was demonstrated on peritoneal exudate and 
bone marrow of the rabbit, granulocytes and 
lymphocytes of man, and peritoneal macrophages, 
kidney and spleen cells of tlie mouse. Special at- 
tention was focused on the peritoneal macrophages 
of mice inasmuch as these are the phagocytic cells 
directly involved in the resistance of this species 
to peritoneally induced experimental infections; 
endotoxin is known to increase resistance to these 
infections. 

Direct exposure in vitro of various cells to high 
concentrations (50 /xg or more/ml) of the most 
potent endotoxins resulted in enhanced glycolysis, 
but no evident injury to the cells. This was not 
always the case in vivo. In mice given endotoxin, 
biphasic responses were clearly apparent in peri- 
toneal macrophages and spleen cells. The level 
of aerobic glycolysis displayed by these cells after 
harvest continued at a constant rate. The cells 
removed from the host did not pass through the 
progressive changes in activity which are found at 
varioiis times in vivo. With small doses of endo- 
toxin only stimulation occurred, but with higher 
doses there was an initial period of depression 
followed by a period of stimulation, which with 
large doses lasted for as long as 8 days. The 
stimulation by endotoxin of glycolytic activity of 
macrophages and spleen cells in vitro was well cor- 
related with the enhanced glycolytic activity of 
these cells harvested from mice that had been in- 
jected with a small dose of endotoxin. In parallel 
metabolic and infection studies, the glycolytic be- 
havior in vitro of mouse peritoneal macrophages 
was correlated with their phagocytic activity in 
vivo. 

Natural Antibodies 

The intriguing nature of natural antibodies was 
illuminated by several lines of study, in which 
various workers collaborated. One study dealt 
with the increase in natural antibodies after ad- 
ministration of endotoxin. The annual report for 
last year described the bactericidal method which 
was then employed to explore the humoral changes 
in mice and rabbits given endotoxin. It was 
found that following the administration of endo- 
toxin to mice, the bactericidal activity of their 
serum for S. typhosa, E. coli., and Six. dysenteriae 
was consistently raised to an activity I14 to 4 



times that found in normal mouse serum. Just as 
had been reported previously in our studies on the 
increase in resistance brought about by endotoxins, 
many factors were found to influence the onset, 
magnitude and duration of these changes. 
Among these factors were the properties of the 
endotoxin itself, the dose, the strain of mice, the 
interval between injection of endotoxin and with- 
drawal of serum samples and the antigenic rela- 
tionship of the endotoxin to the challenge culture. 
Three endotoxins from different sources and with 
individual immunological specificities were effec- 
tive but varied considerably as regards the quan- 
tity required to increase the levels of antibody 
against unrelated Enterobacteriacea. It was 
found repeatedly that, with one hour, the intra- 
venous administration of endotoxin produced a 
substantial increase in antibody levels against im- 
munologically imrelated Gram-negative species. 
The magnitude of this elevation and the duration 
of the enhanced antibody levels appeared to be 
related to the dose of endotoxin. For example, 
the augmented levels following 10-50 /ig persisted 
for 72 to 96 hours, whereas those for 1 and 0.1 yxg 
lasted about 24 and 6 hours, respectively. It is 
significant that, following subsisdence of the en- 
dotoxin effect, there was a return to the original 
levels of antibody. It was found that, within one 
hour after a large dose of endotoxin (50 /j,g), 
bactericidal activity for the homologous strain fell, 
presumably as a result of complexing of endotoxin 
with homologous antibody in the host. During 
this period bactericidal activity for heterologous 
strains was increased markedly, indicating that tlie 
response involved specific antibodies. Further 
e^adence for specificity was pro^aded by absorption 
and inhibition tests with endotoxins of differing 
antigenic specificity. 

Thus, following endotoxin administration there 
occurred a general release of substances active 
against Enterobacteriaceae which show the specific 
activity characteristic of antibodies. These altera- 
tions in the level of antibody were produced with 
endotoxin in amoimts known to modify resistance 
of mice to experimental infection. It is of interest 
that the magnitude and duration of this serum 
effect was dose dependent in a manner analogous 
to the changes in "properdin" levels and resistance 
such as had been reported from this Section 
previously. 



NATIONAL CANCER INSTITUTE 



31 



The capacity to opsonize organisms fox* phago- 
cytosis is a distinctive property of specific anti- 
body. Since these antibodies effectively prepare 
bacteria for the letlial action of complement, it is 
a reasonable assumption that they can also func- 
tion as opsonins. Although no direct correlation 
has been established, these changes are likely to be 
involved in determining the outcome of infection 
since they affect (a) the very cells which are con- 
cerned in host defense against intraperitoneal 
challege and (b) a humoral factor which is known 
to increase the phagocytic efficiency of these cells. 

Exploratory experiments were performed to de- 
termine whether the administration of endotoxin 
affected the levels in serum of natural antibodies 
other than those directed against Gram-negative 
bacteria. Microgram amounts of endotoxin pro- 
duced, in rabbits, a rapid increase in the level of 
natural antibodies against various Enteroiacte- 
riae, in a manner even more pronounced than that 
observed in mice. There also occurred, however, 
analogous increases in levels of natural antibodies 
to foreign erythrocytes and to mouse tmnor cells. 
These findings suggest that the effect of endotoxin 
may well involve a variety of situations where the 
host is naturally and continually subjected to anti- 
genic stimulation. 

There has developed an increasing awareness 
that the characteristic reactivity of manunals to 
endotoxin may be a consequence of the continued 
presence of Gram-negative bacteria in the intes- 
tinal tract from shortly after birth. To determine 
whether the intestinal flora contribute to endotoxin 
hyperreactivity, germ-free and conventionally 
reared mice of similar genetic stock, maintained 
on the same sterilized diet, were compared as re- 
gards their reactions to bacterial endotoxin. No 
significant difference was found as regards tlie 
following: elevation in the levels of antibody to 
Gram-negative bacteria ; stimulation of metabolic 
activity of spleen cells and of peritoneal macro- 
phages; susceptibility to infection with S. ty- 
fhosa; induction by endotoxin of increased resist- 
ance to infection; and LD50 of endotoxin. Tlius 
the presence or absence of a bacterial flora does not 
appear to control the susceptibility of mice to 
endotoxin. 

A study was made of the origin, occurrence, and 
properties of natural antibodies to Gram-negative 



bacteria in the normal serum of several animal 
species. Antibody was measured by a procedure 
described in the preceding amiual report, based on 
the bactericidal reaction carried out under condi- 
tions in which activity was a function of the 
amount of antibody contributed by the test serum. 

Antibodies to representative test strains of 
Salmonella, Shigella, Escherichia, Proteus, Pseu- 
domonas, Serratia, and Aerobacter were demon- 
strated in normal human serum. These antibodies 
were also fomid to be widely distributed in the 
sera of various experimental animals. Absorption 
with homologous bacteria and inhibition of bac- 
tericidal activity with purified homologous so- 
matic antigen affirmed the specific nature of these 
antibodies. Absorption with graded amounts of 
bacterial suspensions showed that a large excess 
of bacteria led to nonspecific removal of antibodies. 
Analogous findings were also made with immune 
antibody. These obsei-vations help to explain how 
earlier workers arrived at the concept of non- 
specificity of natural antibodies reactive with 
Enterobacteriaceae. 

As determined by quantitative absorption tests 
no difference could be fomid in the avidity of 
natural and immmie antibody. Natural antibodies 
of various species were found to be significantly 
less heat stable than immmae antibodies. It has 
been suggested that natural antibody is more heat 
labile than imnaune because it is present in low 
concentration. However, no differences were de- 
tected between the heat liability of diluted and 
undiluted immune serum added to salme or to 
absorbed normal senun. 

The time of appearance of antibodies to S. 
typhosa and E. coli varied in yomig animals of 
different species. Mice developed these anti- 
bodies at the earliest age, with guinea pigs, rats, 
and rabbits following in that order. 

These findings attest to the multiplicity of 
natural antibodies in serum. The ubiquity of 
Gram-negative bacteria makes it inevitable that 
animals may be exposed to them in ways ranging 
from frank or overt infection, commensal status, 
or the simple process of continued ingestion of 
dead bacteria. It seems unlikely, however, that 
the smaller animals, in their comparatively short 
life span, could be exposed to the entire range of 
Gram-negative species. It is more likclv that 



32 



ANTSrXJAL REVIEW OF ESTTRAMURAL RESEARCH 



this category of antigenic determinants is more 
widely distributed than tlae Enterohacteriaceae 
and can be derived from non-bacterial soiu'ces. 

Animals reared in a bacteria-free environment 
lack the antigenic contribution normally derived 
from living mtestinal flora. The level of gamma 
globulin in such animals is known to be reduced, 
but is nonetheless present in significant amounts. 
If living bacteria are important stimulator of 
antibody production to Gram-negative species, 
germ-free animals should have significantly re- 
duced levels. 

Accordingly, serwm from adult germ-free m,ice, 
rats, chickens, rabbits and swine were examined 
for antibody to E. coli and S. tyfhosa. Animals 
of comparable age and genetic stock, maintained 
on the same sterilized diet, but exposed naturally 
to bacterial contamination, sensed as controls. 
Periodic checks indicated that these conventional 
controls consistently harbor various bacterial 
species including Gram-negative serotypes. It 
was found that, as measured by the bactericidal 
test, the levels of antibody in germ-free mice was 
similar to that found in control mice. On the 
other hand, no detectable antibody against E. coli 
or 8. typhosa was found in sera of germ-free rats, 
chickens, rabbits, and swine. In conventional 
controls, however, these antibodies were present in 
substantial concentrations. It is known that im- 
mature animals of different species vary consider- 
ably in their capacity for absorption of intact pro- 
tein from the intestine. If natural antibodies do 
in fact arise as a consequence of antigens absorbed 
from the digestive system, this species difference 
may have special significance. 

Experiments were conducted to obtain infor- 
mation on the stimulation of antibody production 
by antigenic material in the food, particularly 
killed bacteria. Adult germ-free rats, without 
demonstrable antibody to E. coli and S. tyfliosa, 
were fed the usual autoclaved diet in which heat- 
killed S. typhosa had been incorporated to the ex- 
tent of 0.1% on a dry weight basis. Another 
group of Germ-free rats was given a single 
injection i.p. of 100 /^,g typhoid endotoxin. Seriun 
specimens for antibody measurement were col- 
lected at various intervals. Little or none of these 
antibodies was found in the serum of these rats 
during 30 days of ingestion of the killed typhoid 
bacilli. In the rats that received a single injection 



of endotoxin, antibody to S. typhosa was detect- 
able 1 day later and reached high levels within 4 
days. 

Immunological distmctions between normal and 
neoplastic tissues may represent the most subtle of 
the various ways in which they differ. Greater 
understanding of such differences would enhance 
the likelihood of development of more effective 
measures of control. Information on the nature 
of the host's agencies, both humoral and cellular, 
which participate in either natm-al or induced 
immimity, and the manner in whicli they partici- 
pate, is essential for this imderstanding. 

Macromolecular Chemistry Section 

Fundamental studies, under the leadership of 
Dr. P. T. Mora, were carried out on macro- 
molecular interactions in biologic processes, e.g., 
enzyme-substrate and antigen-antibody inter- 
actions, viral infection of cells, etc. Such inter- 
actions were profoundly altered, or prevented 
altogether, by polyelectrolytes both anionic and 
cationic. 



Synthesis and Structure 

To the previously accomplished synthesis of 
anionic polysaccharides, there has been added the 
synthesis of cationic polysaccharides. Basic 
derivatives of the synthetic polyglucoses were pre- 
pared with a very high degree of substitution of 
cationic groups. It is possible to introduce up 
to 1.6 catonic groups per anhydroglucose unit and 
thus synthesize, without degradation of the poly- 
saccharide, a very strongly cationic derivative. 
The best method was the reaction of 1-diethyl- 
amino-2, 3-epoxypropane with polyglucose in 
aqueous sodium carbonate, when the ether deri- 
vative formed with hydroxyls of the polysac- 
charide. The derivative is a tertiary amine. It 
was then easy to convert this tertiai-y amine 
derivative to the corresponding quatemai-y am- 
monium derivative which is even a stronger 
base. 

Work has been carried out aimed at the 
understanding of the st')nictures of synthetic poly- 
saccharides, polypeptides, and proteins. The de- 
tails of the molecular structure are being studied 
by an able group of polysaccharide chemists imder 



NATIONAL CANCER INSTITUTE 



33 



Prof. G. G. S. Dutton at the Chemistry Depart- 
ment, University of British Columbia, Van- 
couver, supported by NIH Grant No. KG7652C1. 
We are helping these studies by providing samples 
of polysaccharides (poly glucose, poly xylose, poly- 
galactose and polyarabinose up to this time) and 
teclinical information from our earlier synthetic 
work and from our macromolecular characteriza- 
tion studies. The knowledge of the fine structure 
will help in correlating structure and immuni- 
logical properties, since these synthetic polysac- 
charides, when the molecular weight is above 
about 200j000 possess antigenic properties. 

Certain structural problems of proteins were 
approached by Dr. S. Sliifrin who was interested 
in checking the claim that the a helix configiiration 
of the polypeptide chains, and interaction of tyro- 
sin and tiyptophane residues, causes the observed 
fluorescence of proteins. For models he chose 
synthetic polypeptides which, from optical rota- 
tory dispersion studies, are said to be 100% in 
the a helix form in dimethylformamide solution 
(Sela). Wlien he compared absorption and fluo- 
rescent properties of the aromatic polyamino acids 
poly-L-tryptophane, poly-DL-tryptophane, poly- 
L-tyrosine and poly-L-phenylalanine with the 
corresponding N-acetyl derivatives, he could not 
find any spectroscopic effect of adjacent aromatic 
residues apart from the fluorescence quenching ex- 
pected from concentration quenching. This in- 
dicates that side group interactions such as in- 
teractions of tryptophane residues cannot be used 
to explain protein fluorescence. In general a more 
careful reinvestigation of the a helix in protein 
and polypeptide structure is in order, since the 
optical rotatory dispersion was used until now 
somewhat indiscriminately to infer helical struc- 
ture. Actually, similar optical rotatory disper- 
sion can result from changes in the solvent struc- 
ture aroimd the protein, and more careful studies 
with fluorescence and other independent methods 
would be helpful to provide fundamental informa- 
tion on the secondary structure of polypeptides 
and proteins. 

Furthermore, fluorescence is an extremely sensi- 
tive method of studying molecular interactions, 
for example enzyme co-enzyme and substrate 
interaction. It is with this in mind that Dr. 
Sliifrin is constructing a sensitive spectroplioto- 
fluorometer, and we expect that such an instru- 



ment would be of great use for many diverse 
studies on macromolecular interaction in this 
Laboratory. 

In another approach to protein and enzyme 
structure he tried to develop a specific and sensi- 
tive fluorescent reagent for combination with the 
sulfhydi-yl groups of proteins and thus develop 
information on the position of these groups in 
the protein chain. The sulf hydryl groups in pro- 
teins are important because they are vei-y reactive 
and, if they are tied up, the enzyme activity is 
usually lost ; thus they probably relate to the active 
center of the enzymes. Also they form S-S cross 
links and stabilize the tertiary structure of pro- 
teins. Dr. Shifrin condensed the highly fluo- 
rescent N, N-dimethylamino-naphthaline-S-sul- 
fonyl chloride with the hj^drazide of isomaleimide, 
the latter chemical being a specific i-eagent which 
in turn condenses with sulfhydryl groups. How- 
ever, because the condensation products of the 
two organic molecules turned out to be unstable, 
this approach was discontinued. 

Enzyme Inhibtion With Poly electrolytes 

In continuing the lead of our earlier work on 
the genex-al phenomena of reversible inhibition of 
enzymes with oppositely charged polyelectrolytes, 
the inhibition of the cationic protein ribonuclease 
with the anionic polyelectrolyte polyglucose sul- 
fate was chosen for detailed examination of pH 
and salt dependence. The inhibition of ENase 
was much more effective at lower pH than at 
higher, when measured at pH values 6, 7 and 8, 
and also in lower salt concentration at any of these 
pH values. This is in line with an explanation 
that electrostatic forces hold together the enzyme 
and the inhibitor polyelectrolyte in a complex, and 
that such forces are the most important causes of 
this type of enzyme inhibition : at lower pH the 
ENase is more cationic, and the charges of the 
macromolecules are less shielded m low ionic 
media. 

Advice and samples of polyglucose sulfates and 
of polyglucose carboxyl derivatives have been 
provided to scores of biochemists who are now 
regularly using these polyanions to inhibit various 
catonic enzymes (ENase, DNase, trypsin, cyto- 
chrom C-oxidase, etc.), following our original 
publications. 



34 



AJSnsrUAL REVIEW OF INTRAMURAL RESEARCH 



Inactivation of 8. marcescens Polysaccharide 

In following up earlier leads, obtained in other 
parts of tliis Laboratory, on detoxification of endo- 
toxic polysaccharides with a system in normal 
blood and a subcellular fraction from liver, a com- 
ponent was leached out of normal liver cells by 
Dr. Oroszlan of higher potency inactivating the 
tumor-necrotizing capability of S. marcescens 
polysaccharide. In the fractionation studies he 
noticed that inactivating potency increased as the 
cationic property of the protein fraction in- 
creased. It became evident that the endotoxic 
polysaccharide, which is an anionic macromole- 
cule, can be inactivated with various kinds of 
cationic macromolecules including our synthetic 
polyglucose amines. Furthermore, such inactiva- 
tion can be reversed by adding a more strongly 
anionic polymer than the bacterial polysaccharide 
(for example by adding polyglucose sulfate) ; the 
bacterial endotoxin then again displays tumor- 
necrotizmg activity. This indicated that the 
mechanism of inactivation of the tumor necrotiz- 
ing potency by these proteins from liver cells was 
through interaction of oppositely charged macro- 
molecules rather than through an enzymatic proc- 
ess. Dr. Orozlan has also been studying the 
macromolecular properties of the endotoxic poly- 
saccharide preparation from S. marcescens, with 
the purpose of separating the most active com- 
ponent from the relatively inert components still 
present. 

Bacieriophage 

The concept of inhibition of various kinds of 
biological activity by means of complexing the 
active polymers with oppositely charged poly- 
electrolytes was extended to the demonstration of 
reversible hloching of an antibody. In the anti- 
serum against T2 bacteriophage the antibody ac- 
tivity can be blocked with a strongly charged 
poly electrolyte (for example, with the anionic 
f)olyglucose sulfate) but the antibody recovers its 
activity if there is added to this system an op- 
positely charged polyelectrolyte to that employed 
first (for example, the cationic spermidine). 
Higliest reactivation occurs when the second poly- 
electrolyte is added in stoichiometrically equiva- 
lent amount to the first polyelectrolyte, just suffi- 
cient to neutralize the charges. Thus it was pos- 
sible to inactivate and to reactivate the T*2 bac- 



teriophage antisei-um in a predictable and con- 
trolled way. Our explanation for the inactiva- 
tion of the antibodj' was that the strong polyelec- 
trolyte added first (anionic polyelectrolytes are 
generally more effective, but cationic polyelectro- 
lytes also show this effect) complexed with the 
amphoteric antibody jDrotein; in the complex tlie 
antibody was unable to combine effectively with 
the virus. When a second strong polyelectrolyte, 
oppositely charged to the first polyelectrolyte, was 
added, a preferential complex formed between 
these two, and the liberated antibody was now 
free to combine with and neutralize the virus. 
Analogous research on better defined systems 
(using isolated antibody and antigen) would un- 
doubtedly lead to better understanding of the 
forces which bring about the antibody-antigen in- 
teraction. Also, our results suggest new methods 
for the fractionation of the antibody from the 
antiserum with the help of polyelectrolytes. 
Finally, it gave some leads on how to influence 
(block and reactivate in a controlled way) anti- 
sei'um activity against a virus in vitro. 

Earlier work in this Section had shown that in- 
cubation of T2 phage with anionic polyelectrolytes 
abolished the ability of the phage to reproduce in 
E. coli cells without destroying phage structure. 
Dr. Rizvi gained further insight into this process. 
He developed new methods to inactivate high con- 
centrations of T2 bacteriophage (10^-phage/ral) 
with polyglucose sulfate (1 mg/ml) without any 
concomittant release of T2 DNA from inside the 
protein coat. He achieved this by using properly 
selected buffers with the polyglucose sulfate ; thus 
the phage was not exposed to local extremes of 
pH. Dr. Rizvi currently is extending the poly- 
anion inhibition of the T2 bacteriophage into the 
whole T series of bacteriophages from Tl to T7, 
on a large scale, with special attention to tlie T-i d 
mutant which has a protein coat penneable to low 
molecular compounds and can be prepared Avith- 
out the internal cations putrescine or spermidine. 
Tlie cations putrescine and spermidine, found 
recently by B. Ames of NIAMD to be j^resent 
inside of the protein coat of certain of the bac- 
teriophages, have an important role in neutralizing 
the anionic charges of the DNA. 

Our polyanion treatment undoubtedly causes 
some increase to the permeability of the protein 
coat. Dr. Rizvi observed that the percent in- 



NATIONAL CANCER INSTITUTE 



35 



activation of bacteriophage viability after the 
polyanion treatment is the same as the percent 
loss of the internal low molecular weight cations 
putrescine and spermidine, released by this treat- 
ment through the protein coat from inside the 
phage. Polyglucose sulfate apparently competes 
with the DNA and preferentially complexes with 
these cations. The loss of the cations must dis- 
turb the original structure of the DNA in the head 
of the bacteriophage ; with the permeability change 
of the protein coat, this appears to be the main 
cause of the loss of viral infectivity. 

Virus Inactivation With Poly electrolytes 

The cationic polyglucose derivatives prepared in 
this Laboratory were examined by Prof. A. Di 
Marco in Milan, Italy, for their effect on influenza 
virus (strain A2/W29). In very low concentra- 
tions these polymers inliibited the hemaggluti- 
nating action of the virus; they also reduced 
somewhat the mortality of mice when infected 
intranasally with this virus previously incubated 
with the cationic polyglucose amines. Polyglucose 
amines themselves showed hemoagglutinant ac- 
tion at higher concentration : this action decreased 
when such solutions were kept incubated at 37° C 
with chorio-allantoic membrane. Apparently 
this hemoagglutinant action of the polyglucose 
ammes were exerted at sites different from those 
which the influenza virus uses to agglutinate red 
cells, because when erythrocytes were incubated 
first with influenza virus and the virus was eluted, 
then these erytlirocytes still agglutinated with the 
polyglucose amines. 

Cationic polyelectrolytes also inhibit the bac- 
teriophages. Our work showed that this inhibi- 
tion is strongest in about 0.9% saline, and de- 
creases both above and below this salt concentra- 
tion. This mdicates that other than purely 
electrostatic factors must participate in such 
inactivation. 

Viability of the mouse infectious encephalomye- 
litis virus was found by Drs. K. K. Takemato and 
H. Liehaber of NIAID to be reduced by a direct 
interaction with polyglucose sulfate. They also 
found that this anionic polymer affected plaque 
morphology in a fashion similar to that of a sul- 
fated polysaccharide present in small concentra- 
tion in the agar usually employed in the overlay 
during the assay of these viruses. 



The charged iDolysaccharide derivatives syn- 
thesized by J. W. Wood continued to be m great 
demand by biochemists working on enzyme and 
on virus mhibition studies, both in this country 
and abroad. 

Biochemical Pharmacology Section 

Dr. Goldin and his group have continued to 
investigate a considerable niunber of facets in the 
field of basic science chemotherapy. Their objec- 
tives have been: chemotherapy studies of antitu- 
mor and antiviral agents and the mechanism of 
their actions; synthesis of new agents; study of 
host-tumor relationships with respect to drug 
action ; and establislunent of new therapeutic prin- 
ciples and procedures in the management of tumor 
growth in animals. 

Many aspects of the program were the product 
of collaborative work with investigators in other 
IDarts of the NCI and in institutions elsewhere. 
Close working relationships were maintained with 
clinical colleagues active in chemotherapy investi- 
gations in patients. 

Immunity 

It had been noted in previous work in this Sec- 
tion that, after treatment of advanced leukemia 
1210 in Fi hybrids of CxDBA with halogenated 
derivatives of amethopterin, an appreciable num- 
ber of mice survived. Many of them were found 
to be immune to a second inoculation of the sensi- 
tive subline of L-1210, and also to antif olic-resist- 
ant sublines. It was than found that this host 
immunity could aid in the chemotherapy of the 
amethopterin-resistant sublines. 

These observations were followed up in a num- 
ber of ways. Immunization of hybrid mice with 
normal spleen, x-irradiated normal spleen, and 
x-iri'adiated leukemic spleen, followed by chal- 
lenge with L-1210 or skin grafts, suggested that 
L-1210, which originated in DBA mice, possesses 
some antigenicity for the CxDBA hybrid. 

Siiace leukemia L1210 is a transplantable tumor 
with a long laboratory history, it is not unexpected 
that it possesses some slight incompatability to 
the isologous host. "Without therapy this incom- 
patability is masked by the rapid growth of the 
tmnor and early death of the animal. Therapy 
with highly effective drugs such as the halogenated 



36 



AJSnSTTJAL REVIEW OF INTRAMTJRAL RESEARCH 



derivatives of aniethopterin, by holding the tumor 
growth in check, apparently permits sufficient time 
for the host to respond to the weak antigenic stim- 
ulus. The immune response in turn is added to the 
direct antitumor action of the agent, resulting in 
extensive suiwival times and cures. 

It is of considerable interest that therapy of 
leukemia L1210 in DBA/2 mice is less successful 
than in CXDBA Fi hybrids. In DBA mice, 
halogenated derivatives of amethopterin pro- 
longed survival time but failed to achieve "cures". 
It had been considered previously that this was 
attributable to lower drug tolerance of the DBA 
mice. Evidence has now been obtained that the 
DBA mice are less responsive to immunization 
than the hybrid mice. Investigations are in prog- 
ress on the nature of this unusual observation and 
its possible relationship to the gi'eater resistance 
to therapy of the leukemia in DBA mice, the strain 
of origin. 

Increases in survival time were obtained in mice 
inoculated intracerebrally or subcutaneously with 
FR-3, an antifolic-resistant subline of L1210, on 
treatment with 3'-bromo-5'-chloromethopterin 
(BCM) if the mice were first treated with BCM 
for intracerebral inoculation of sensitive L1210. 

The series of experiments on the effect of surgi- 
cal adjuvant therapy reported last year has been 
extended and completed. It had been demon- 
strated that a combination of surgery and chemo- 
therapy (6-mercaptopurine) was more effective 
than surgery alone or chemotherapy alone in in- 
creasing the lifespan of mice bearing advanced 
adenocarcinoma Ca-YSS. Complete remission of 
tumor occurred in a high percentage of cases fol- 
lowing surgery and adjuvant therapy. Studies 
have been conducted showing that host immrmity 
contributes to the therapeutic response to 6-MP. 
Survivors of adenocarcinoma 755 resulting from 
successful surgery plus treatment with 6-MP 
showed immunity to reinoculation. This immun- 
ity was shown to occur relatively early during the 
course of therapy. Also, mice inoculated intra- 
peritoneally with x-irradiated carcinoma 755 were 
higlily refractory to a challenge with viable car- 
cinoma 755. 

Without surgery, in the presence of a large tu- 
mor mass, the moderate immune response to car- 
cinoma 755 apparently does not augment therapy 
sufficiently to permit extensive tumor remission. 



However, when a large part of the tumor is surgi- 
cally enucleated, the combination of chemotherapy 
and immune response is sufficient to result in ex- 
tensive regression and a liigh percentage of 
survivors. 

It had prevously been found in this Section that 
several strains of L1210 elicited a typical homo- 
graft response in a variety of lines of mice and 
that the homograft response was readily sup- 
pressed by treatment with folic acid antagonists. 
Extension of these findmgs showed that variation 
in the size of the inocidmn can alter the homoffraf t 
response. With progressive dilution of the inocu- 
lum in out-of-strain mice, the proportion of tumor 
takes terminating in death of the animals in- 
creased mai-kedly. This phenomenon was des- 
ignated as "the dilution effect". 

Antifolic therapy of the resistant subline re- 
sulted in progressive growth of the leukemia and 
death over the entire range of inocuhun concen- 
trations, thereby removmg the dilution effect. 
Simultaneous admmistration of the metabolite, 
citrovorum factor, prevented the abrogation of the 
homograft response by amethopterin, and m es- 
sence, restored the dilution effect. 

Concomitant injection of normal tissue (spleen 
and blood) or x-irradiated leuliemic tissue pre- 
vented the lethality obsei-ved with low concen- 
trations of leukemic inocula, thereby also remov- 
ing the dilution effect. 

Suppression of host immunity, rather than the 
dihotion effect would appear to accomit for the ob- 
servation that therapy of a resistant tmnor abro- 
gates the homograft response. However, the 
dilution effect would appear to be an important 
phenomenon in relation to screening and drug 
evaluation with transplantable timiors. 

The growing interest in the extent to which 
antitumor agents suppress host immune reactions 
had led to the development of a model system for 
the evaluation of such antihost effects of cliemo- 
therapeutic agents. Previously, we had reported 
that therapy of a resistant tumor abrogated the 
homograft reaction. In that system, however, 
therapy of the tumorous animal complicated the 
evaluation of the anti-host effects of the com- 
pounds. 

The current system measures the extent of 
growth of leukemia L1210 in homologous mice fol- 
lowing pretreatment with the candidate drug. 



NATIONAL CANCER INSTITUTE 



37 



The system avoids any direct effect of the drug on 
the tumor, thereby permittmg a critical evaluation 
of the effect of candidate compounds on the im- 
mime response of the host. To date, sarcolysin, 
triethylene melamine, Cytoxan, amethopterin, and 
6-mercaptopurine have been tested in this system, 
employing leukemia L1210 in C57B1/6 mice. The 
alkylating agents were quite effective, with sar- 
colysin producing the most extensive suppression 
of host immunity. 6-Mercaptopurine was less ef- 
fective, and amethopterin was relatively ineffective 
in tliis system. Homograft suppression, as evi- 
dence by an increased incidence of takes, was ap- 
parent only at toxic levels with these agents, and 
then was not extensive. 

In other experiments the system described above 
was modified to include both skin and tumor 
homografts. To date, a good correlation lias been 
observed between survival of skin and tumor 
homografts in control animals, m which both are 
rejected, and in animals whose immmiological re- 
sponse has been impaired by drug treatment. 
BALB/c animals pretreated with sarcolysin and 
subsequently implanted with leukemia, died of 
progressive tumor growth. In comparable groups 
of pretreated animals, the median survival time 
of DBA skin grafts was approximately twice the 
median sui^vival time of the tumorous animals. In 
contrast, on pretreatment with amethopterin, sur- 
vival of skin graft and tmnor approximated that 
of the respective controls. 

Continuous daily post treatment with an optimal 
dose of amethopterin abrogated the homograft re- 
sponse to the tumor, and tlie animals all suc- 
cumbed. In comparably treated animals the skin 
homograft reaction occurred, but graft survival 
was increased approximately 50 percent over the 
median survival time of tumor animals. 

Vi'TU'S Leuk&mia 

Experunents have been carried out in vivo with 
the Maloney virus and with the Kauscher virus. 

Mice bearing transplants of whole-cell leukemia 
generated by the Maloney virus survived longer on 
treatment with alkylating agents such as Cytoxan, 
TEM, and sarcolysin than with antifolics, purine- 
and pyrimidine-antagonists, or antibiotics. The 
several lines of transplantable virus leukemia that 
have been established showed a wide range of drug 
sensitivity, e.g., to Cytoxan. These observations 



indicate that a population of mice m- wMch the 
Maloney virus liad uiduced lymphocytic leukemia 
would jDrobably show the broad range of sensi- 
tivity to drugs characteristically seen in clinical 
leukemia. 

One of the whole-cell lines was quite sensitive 
to therapy with several alkylatmg agents and 
x-irradiation. Following apparently successful 
therapy of this transplanted whole-cell line, ani- 
mals subsequently succumbed, frequently after 100 
days. Pathologic examination of some of these 
leukemic long-term survivors, by Dr. Thelma 
Dunn, revealed a widely disseminated lymphocytic 
neoplasm. 

Since the transplant inoculum contained both 
leukemic tisue and tlie inducing virus, it was of 
fundamental importance to determine whether the 
animals were succumbing to slow-growing vari- 
ants in the transplanted cell population, or to a pri- 
mary leukemia, induced by the virus. Data 
obtained from transplantability studies with tissue 
obtamed from these long term survivors strongly 
suggest that the agents (TEM, Cytoxan, sarcolysin 
and x-irradiation) eradicated the whole-cell dis- 
ease, and that the virus in the initial leukemic im- 
plant subsequently induced a second lymphocytic 
neoplasm in these animals. 

The system described above, in which thei'apy 
induced a remission, which was followed by sub- 
sequent reappearance of leukemia, appears to ap- 
proximate closely the clinical experience observed 
with acute lymphocytic leukemia in children. 
Thus, this system provides an experimental tool 
for investigating maintenance of remission and 
"mduced" resistance. In addition, it raises the 
question as to a possible role of virus as an inciter 
of human leukemia. 

Cytoxan, 5-fluorouracil, and TEM, in pre- 
liminary experiments, produced a decrease in 
spleen size in mice bearing the Eausclier viras. 

CNS Tumors 

Cytoxan was fomid to be much less eft'ective in 
the treatment of intracerebral L1210 tlian of the 
subcutaneous. This finding was obtained both on 
intracranial injection of the drug as well as with 
subcutaneous. 

The relative effectiveness of a series of drugs 
against L1210 inoculated I.C. or S.C. was ascer- 
tained with a single S.C. injection of the maxi- 



38 



ANlSrCTAL REVIEW OF INTRAMTJRAL RESEARCH 



mum tolerated dose of the drug 24 hours after 
implantation of the leukemia. All the drugs 
tested were less effective against I.C. than S.C. 
leukemia. Cytoxan was the most effective against 
both sites. 

The duration of effective drug levels was deter- 
mined by giving a single dose of the drug at vari- 
ous intervals prior to inoculation of leukemia I.C. 
or S.C. Chlorambucil was ineffective. The 
others (Cytoxan, TEM, and L-Sarcolysin) were 
more effective against the S.C. than I.C. leukemia. 
The effective dose level, however, is maintained 
for only a brief period — inhibition was obtained 
when the interval between the drug and tumor 
administration was one-half an hour, but pre- 
injection of the drug one hour prior to inocula- 
tion of tumor was without effect. 

Sequestration of tumor cells in the brain, and 
less efficient entry of drug into the brain site, 
have important bearing on refractoriness to com- 
plete remission in chemotherapy. Detailed patho- 
logic studies of these mice showed features similar 
to those in the brain and meninges of patients 
who died of acute leukemia. 

Systemic chemotherapy of I.C. leukemia with 
Cytoxan apparently destroyed all, or most, of 
the leukemic cells in the spleen, bone marrow and 
dura, but had little, if any, effect on the infiltrate 
of leukemic cells in the arachnoid space. Pro- 
gressive growth of leukemic cells in the arach- 
noidal and perivascular space occurred while ani- 
mals were receiving daily subcutaneous doses of 
Cytoxan. 

Evaluation of Antitumor Agents 

Assay procedures which had been developed in 
this Section were employed in the evaluation of 
new compounds of clinical uiterest. The tumors 
used in the assays were L1210 and resistant vari- 
ants. Sarcomas 37 and 180, carcinoma 755, and 
the Ehrlich ascites tumor. These studies were 
carried out primarily in the Drug Development 
and Evaluation Program (Contract No. SA-43- 
ph-237l). This line of work is fully presented 
in a separate report (NCI 216), but a few of the 
major points may be mentioned here. 

During the past year 67 compounds were 
examined for ability to increase the lifespan of 
mice with systemic L1210. This brought the 
total to over 300 compounds. Sunamary data on 



235 compounds were included in a comprehensive 
"Manual" prepared in collaboration with Dr. 
Howard E. Skipper of Birmingham and Dr. 
Leon Schmidt of Cincinnati. The most active 
compound was 2-chloro-4:'-di-2-imidazo]in-2-yl- 
terephthol-anilide (NSC 38,280). Of the alkyl- 
ating agents, none was more effective than 
Cytoxan. In general, alkylating agents of the 
ethylene-imine type were more effective than ni- 
trogen mustards, methane sulfonates, or epoxides. 
No purine or pyrimidine derivative was more ef- 
fective than 6-MP. Of the antibiotic materials, 
Duazomycin A exhibited 1/3 to i/4 the activity of 
MTX, but showed no significant advantage over 
the structurally -related azaserine. 

For a series of compounds, variations in the 
treatment schedules were investigated. The rela- 
tive advantages of drug administration daily, 
every 2 days, and every 4 days were compared 
with the effectiveness of a single treatment. Each 
drug displayed its own characteristics in this 
regard. 

Combination therapy with two drugs was found 
to be more effective than with either alone in the 
case of the following pairs: NSC 38,280 plus 
MTX; Cytoxan plus MTX; and Cytoxan plus 
6-MP. In contrast, combination of MTX plus 
6-aminonicotinamide was no more effective than 
MTX alone. 

Of the 17 additional compoimds evaluated 
against Ehi'lich ascites tumor, none produced a 
greater survival time than previously obtained 
in this Laboratoi-y with N-methyl-formaniide. 
As regards the newer data on increased time of 
survival of mice bearing the three solid tumors, 
alanine nitrogen mustard continued to be the most 
effective for S37 and SlSO; Ca755 has been ])ur- 
ticularly sensitive to many purine derivatives and 
to Cytoxan. 

A comprehensive study of the relationship be- 
tAveen inhibition of the local tumor and increase 
in survival time elicited by the treatment of mice 
with S37 or Ca-755 was conducted. No necessary 
correlation between inhibition of tlie growth of 
the local tumor and increased survival time was 
found. For example, when mice bearing early 
Ca-755 were treated, daily to death, with Cj'toxan 
or 6-MP, both compounds provided a 100 percent 
increase in median survival time. With Cytoxan, 
the increase in survival time was accompanied by 



NATIONAL CANCER INSTITUTE 



39 



only a moderate inliibition of the local tumor. 
The equivalent increase in survival time produced 
by 6-MP was accompanied by a marked inhibition 
of the local tumor. Doses of Cytoxan which 
caused marked tumor uiliibition resulted in de- 
creased survival times because of their toxicity for 
the host. A- 139 produced a high degree of tumor 
inhibition in mice with early Ca-755, but failed 
to uacrease survival time. N-Methylformamide 
increased the survival time of mice with early 
S-37 at dosage levels which are not markedly in- 
hibitory to the local tumor. 6-Thioguanine pro- 
duced a high degree of tumor inhibition of S-37 
but failed to increase the survival time of the mice. 
In general, these studies showed that drug efficacy 
in increasing survival time may be accompanied 
by a greater or lesser degree of tumor inliibition. 

Collaborative work has been m progress with 
Prof. Orrie Friedman of Brandeis Univ., who is 
synthesizing new compounds related to Cytoxan 
and other types of mustards with carrier groups. 
Cytoxyl alcohol, a possible metabolic product of 
Cytoxan, was markedly less active against L1210. 
Arrangements were made with Dr. G. M. Timmis 
of the Chester Beatty Institute in London to in- 
vestigate here the antitumor effects of selected 
compounds synthesized there. 

Folic Acid Antagonists: Mechanisms 

Prefolic A, both naturally occurring and syn- 
thetic specimens, was f oimd to be active as a meta- 
bolite in the L1210 system. (Drs. Keresztesy and 
Donaldson, NIAMD had isolated prefolic A from 
liver, then synthesized 5-methyltetrahydrofolate, 
and showed them to be identical) . It was found 
to be as effective as citrovorum factor in reducing 
the toxicity of amethopterin in mice and m re- 
ducing its antileukemic effect. The N5-ethyl 
and N5-propyl derivatives, prepared by Dr. Ker- 
esztesy, were tested for activity against early 
L1210; neither compound produced prolongation 
of survival. 

Our previoiis observations on the metabolite 
activity of dihydrofolic acid, in which it was 
shown that this compoimd had biological activity 
in the presence of amethopterin similar to fully 
reduced derivatives of folic acid are now being 
extended to an in vivo assay, in collaboration with 
Dr. P. Condit, Oklahoma City. This system 
measures directly the ability of animals, treated 



with amethopterin, to convei't folate or dihydro- 
folate to tetrahydrofolate derivatives. Prelimi- 
nary results have shown that amethopterin at low 
doses will completely inhibit the conversion of 
folate to tetrahydrofolate, but that a dose as high 
as 500 mg/kg of amethopterin will not inliibit the 
conversion of dihydrofolate to tetrahydrofolate. 
This is not in agreement with in vitro studies of 
the reduction of folic acid in which the reduction 
of folate and dihydrofolate are equally sensitive 
to inhibition by amethopterin. It raises the ques- 
tion as to whether the enzyme systems which 
catalyse this reduction in vivo are the same for 
folate and dihydrofolate. 

With Prof. M. Friedkm (Tufts Univ.) we have 
shown that in some antifolate resistant sublines of 
leukemia L1210 there is a considerable increase in 
the level of the enzyme dihydrofolate reductase. 
This led to the suggestion that it might be possi- 
ble to utilize this property of the resistant vai'ients 
to achieve a lethal synthesis of tetrahydrofolate 
analogs by introducing a substrate which would 
be preferentially reduced by the tumor cells with 
high reductase levels. In preliminary woi-k, sev- 
eral simple derivatives of folic acid have not 
shown significant activity. A series of antifolic 
analogs is being synthesized to test for lethal 
synthesis in leukemic variants with high content 
of dihydrofolate reductase. 

We have observed that the toxicity and anti- 
leukemic effect of amethopterm, in mice, produced 
by daily treatment can be reversed by dihydro- 
folic, but not by folic acid. With Dr. Condit, we 
have shown that conversion of folic acid to citro- 
vorum factor in mouse liver is inliibited by small 
doses of amethopterin, while the conversion of 
dihydrofolic acid to citrovoxiim factor is not. To 
explam this behavior, Drs. Schrecker and Mead 
have carried out in vitro studies on the kinetics 
of inhibition of diliydrofolate reductase. The 
enzyme was obtained from a highly resistant sub- 
line of leukemia L1210 with 80-fold increase in 
dihydrofolate reductase activity. It was found 
that this enzyme catalyzed the reduction of both 
folic and dihydrofolic acid at pH 5, but that only 
dihydrofolic acid was reduced at pH 7. At pH 5, 
the reduction of both folic and dihydrofolic acid 
was inliibited irreversibly by amethopterm and 
3,' 5'-dicliloroamethopterin. On a molar basis, 
both antagonists were equally effective inhibitors. 



40 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



This was in agreement with previous findings ob- 
tained by Zakrewski, Werklieiser and Nichol, who 
used an enzyme isolated from chicken liver. At 
pH 7, on the other hand, both amethopterin and 
dichloroamethopterin were found to be non-com- 
petitive inhibitors of dihydrofolate reduction, i.e. 
dUiydrofolic acid was able to reverse partly the 
inliibitory effects of the antagonists. At this pH, 
dichloroamethopterin was a more effective inhibi- 
tor than amethopterin. These findings agree with 
previous findings of Huennekens, Bertino and 
Friedkin. An. amount of inhibitor that com- 
pletely represses the reduction of folic and of 
dihydrof olic acid at pH 5 was f oimd insufficient to 
suppress the reduction of dihydrofolic acid at 
pH 7. These findings may help explain the re- 
versal of amethopterin-induced toxicity and anti- 
leukemic effect by dihydrofolic acid, but not by 
folic acid. 



Drug Resistance 

The development of resistance to a drug to 
which the tumor had been initially responsive is a 
phenomenon of such importance that considerable 
attention was devoted to experimental work on 
this subject. Many variants of L1210 were devel- 
oped in which each subline was resistant to a par- 
ticular agent. Examination of the responses to 
other agents revealed no generalization regarding 
cross resistance, for each variant was a special 
stoiy in itself. The following findings exem- 
plify the results in this line of work. 

Seventeen compounds were tested for their ac- 
tivity in increasing the survival time of mice with 
the 3', 5 '-dichloroamethopterin resistant variant, 
L1210/M663R. This subline of L1210 was cross 
resistant to MTX, but did not display cross re- 
sistance to Cytoxan, NSC 38,280, 6-MP, 6-MP- 
riboside, tetramin, methylglyoxalbisguanylhydra- 
zone, 1-aminocyclopentane carboxylic acid, thio- 
TEPA, TEM, 2-amino-l,3,4-thiadiazole, 5-fluoro- 
uracil, 5-fluorodeoxyuridine, streptovitacin A, 
actinomycin D, 4-aminopyrazolo(3,4-d)pyrimi- 
dine, or 6-aminonicotinamide. 

The L1210/C95 variant of leukemia L1210 arose 
from successive treatment with MTX, 6-MP, and 
Cytoxan. This subline is markedly resistant to 
6-MP and MTX and displays a high degree of 
resistance to Cytoxan. The L1210/C95 variant 



displayed no resistance to methylglyoxalbisguan- 
ylhydrazone or NSC 38,280. 

6-Mercaptopurine (6-MP) and 6-mercaptopu- 
rine riboside (6-MP-Il) were compared for their 
effectiveness against the L1210/AgE subline of 
leukemia L1210. L1210/AgR is resistant to 8-aza- 
g-iianine and has displa^^ed cross-resistance to 
6-MP. The studies showed that L1210/AgR was 
equally resistant to 6-MP and6-MP-R. 

In collaboration with Dr. D. Hutchison and 
J. Biedler of the Sloan-Kettering Institute, a 
number of resistant L1210 sublines were investi- 
gated as regards certain genetic and biochenaical 
factors, viz., the chromosome complement of the 
leukemic cells and their specific dihydrofolate re- 
ductase activity were determined at several trans- 
plant generations. 

Cells of the sensitive parent line possess a sub- 
metacentric chromosome. A mercaptopurine 
(MP) resistant subline which was not collaterally 
resistant to amethopterin, still possessed the sub- 
metacentric chromosome, and had the diliydro- 
folate reductase activity of the parent line. An 
amethopterin-resistant subline had lost the sub- 
metacentric chromosome, and its reductase activ- 
ity was increased 11-fold. Another subline, 
treated serially with a combination of amethop- 
terin, MP, and fluorouracil was resistant to these 
drugs singly and in combination. During the first 
56 transplant generations, it still had the submeta- 
centric chromosome, and its dihydrofolate reduc- 
tase activity was that of the parent line. Between 
the 56th and the 63rd transplant generations, a 
mutation apparently occurred, leading to loss of 
the submetacentric chromosome and an 18-fold in- 
crease in dihydrofolate reductase activity. These 
findings suggest a possible correlation between 
loss of the submetacentric chromosome and in- 
creased dihydrofolate reductase actiiaty. 

The biochemical basis of drug resistance to leu- 
kejnia was investigated by Drs. Schrecker, Mead 
and collaborators with regard to: (a) correlation 
of dihydrofolate reductase activity with increas- 
ing degree of antifolate resistance in mouse leu- 
kemia; (b) correlation between inhibition of 
dihydrofolate reductase and of purine biosynthe- 
sis in leukemic tissues by folic acid antagonists; 
and (c) use of a computer to explore the use of 
dihydrofolate reductase as a tracer of amethopter- 
in-resistant leukemic cells. 



NATIONAL CANCER INSTITUTE 



41 



When L1210 Wcos made resistant to amethop- 
terin, the enzyme activity increased 11-fold. Made 
this variant resistant also to a second antifol 
(dichloroamethopterin) resulted iii a 40- fold m- 
crease. Wlien the latter compound was used alone, 
a variant developed with partial resistance to tliis 
antifol, but there was no concomitant rise in this 
reductase. 

A sublme of L1210 leukemia had previously 
been developed by serial transplantation and treat- 
ment with aniethopterin for 10 generations, fol- 
lowed by similar treatment with dichloroamethop- 
terin for 10 generations. This subline was 
biologically fully resistant to the two drugs. Im- 
mediately following establisliment of this subline 
(FE-8), its dihydrofolic reductase activity was 
increased 80-fold. After 11 transplant genera- 
tions in the absence of treatment, dihydrofolic 
reductase activity was still 60-fold that of the 
parent line, but fell to a 25-fold level at genera- 
tion 14. At the 29th and 33rd transplant genera- 
tion ui the absence of treatment, dihydrofolate 
reductase activity was the same as in the sensitive 
parent line, although the tumor was still resistant 
to amethopterin and dicliloroamethopterin. 
Treatment with 75 mg/kg/day of dichloroame- 
thopterin durmg one generation after 13 or 29 
untreated transplant generations restored the max- 
imal (80-fold) dihydrofolic reductase activity. 
This would be consistent with a mechanism of 
enzyme or with elimination of sensitive cells pro- 
duced by partial reversion of the resistant subline 
to sensitivity. Purine biosynthesis in vivo was 
measured concurrently in several transplant gen- 
erations by means of determining the incorpora- 
tion of radioactive formate into the adenine of 
leukemic spleen and tmnor in the absence and the 
presence of amethopterin or dicliloroamethopterin. 
Dose-response curves were obtained. It was found 
that, as the dihydrofolate reductase activity was 
increased in the leukemic tissues, increased 
amounts of drug were required to produce the same 
inhibition. Wlien dihydrofolate reductase activity 
was increased 80- fold, no inliibition of purine bio- 
synthesis was observed at all, even with very high 
doses. This would suggest that the cells contained 
enzyme in excess of the amount that could be in- 
hibited by the maximal drag concentration capable 
of being established inside the cells. 

Mathematical models were employed in aii at- 



tempt to relate the interplay between such factors 
as life span of a leukemic animal, how early drug 
treatment is initiated, the survival of sensitive and 
resistant leukemic cells in treated animals, the 
incidence of mutation from the sensitive state to 
the resistant state, and the dihydrofolate reductase 
levels in mixed populations of sensitive and resist- 
ant cells. A computer was used to generate theo- 
retical mixed populations of resistant and sensi- 
tive leukemic cells during three simulated passages 
in drug-treated animals. This was done so as to 
mimic the actual enzyme data collected during the 
development of an amethopterin-resistant subline. 
It was concluded that dihydrofolate reductase can 
be used as a meaningful biochemical marker ui 
studies of emerging antifolate-resistant leukemic 
cells. 

A model experiment was conducted by S. Hum- 
phreys and collaborators to determine whether 
resistant leukemic cells with high levels of di- 
hydrofolate reductase might serve as biochemical 
markers, reflecting the efficacy of therapy. This 
was considered feasible since the high levels of 
dihydrofolate reductase could readily be measured 
in tissues showing any extensive degree of inva- 
sion by leukemic cells. In the model experiment, 
the high diliydrof olate resistant leukema was inoc- 
ulated intracerebrally ; treatment was with 
Cytoxan. 

A close correlation was foimd of biochemical, 
biological, and pathological observations. Cy- 
toxan was effective in increasing survival time. 
The systemic disease was held in check by treat- 
ment, as shown by the low level of dihydrofolate 
reductase activity in the spleen and the reduced 
transplantability of spleen suspension. Follow- 
ing therapj^, pathologic examination of the spleen, 
liver, bone marrow, and other extracranial tissues 
revealed little evidence of leukemia. There was 
much more difficulty in holding the disease in 
check in the brain. Tliis, again, was reflected in 
the progressive increase in dihydrofolate reckictase 
m the brain, the success of retransplant, and the 
infiltration of the brain seen on pathologic exami- 
nation. 

Enzymatic markers of this type may be useful in 
studies of fate and distribution of di'ugs, and of 
the blood-brain barrier. They may be employed 
in investigations of tumor invasiveness, and the 
origin of resistance and the efficacy of therapy. 



42 



ANNUAL REVIEW OF INTPIAMUEAL RESEARCH 



High diliyclrof olate reductase in antif olic resistant 
leukemia can serve as a marker which, in conjunc- 
tion with the biological studies and the pathology, 
may provide a means for detailed investigation of 
chemotherapeutic agents. 

Otlier Topics 

In collaboration with Prof. N". O. Kaplan of 
Brandeis Univ., an extensive series of pyridine 
derivatives was studied with regard to ability to 
function as metabolites like nicotuiamide, or as 
antimetabolities like 3-acetylpyridine. The large 
amount of data has been summarized in a com- 
prehensive review which provides a basis for cor- 
relation of the foregmg activities with ability to 
stimulate DPN synthesis in vivo and to form ana- 
logs of DPN by exchange with its nicotinamide 
moiety. 

Dr. Chirigos has been investigating the trans- 
port of amino acids, notably tyrosine, in S37 ascites 
cells. The uptake of L-tyrosine was rapid : an in- 
tracellular concentration nearly 7 times that in the 
external medium was accomplished in less than 
one-half hour. Little sterospecificity was ob- 
served: D-tyrosme, DL-tyrosine and CHa-tryro- 
sine were concentrated nearly as well as the a-f orm. 
Tyramine and p-hydroxyphenylacetic acid were 
not concentrated. Heating at 60° C in buffer, and 
treatment with a variety of metabolic inhibitors 
such as sodium azide and 2,4-dinotrophenol in- 
hibited tyrosine transport. 

During a study of the influence of Sarcolysin 
(phenylalanine mustard) on tumor growth ; it was 
observed the amino acid analogue p-fluorophenyla- 
lanine emitted appreciable fluorescence in the 
ultra-violet. The fluorescence characteristics of 
the positional isomers of other halogenated 
phenylalanines were therefore investigated by Dr. 
Chirigos. It was concluded that fluorine, uniquely 
among the halogens, enhances the fluorescence of 
aromatic rings. 

Much labor, space, animals, and expense may be 
conserved in the continuous maintenance of stocks 
of transplantable tumors by preserving them in 
the frozen state and then thawing them only when 
required for new experiments. The viability, and 
the resistance to chemical agents, of variants of 
L1210 were found to be satisfactorily preserved by 
the storage of frozen tumor cell suspensions. 



Three sublines of L1210 (M46E, AgR, and 
M663E) , after freezing and thawing, yielded com- 
parable reactions to antitumor agents as they did 
when maintained in continuous passage in ani- 
mals. A variety of other tumors, notably sar- 
comas and carcinomas, which had been carried in 
live passage in other parts of this Laboratory have 
now also been preserved in the frozen state. 



DERMATOLOGY BRANCH 

The research activities of the Dermatology Branch 
concern two major areas: (1) Study of normal 
and abnormal growth and differentiation of the 
epidermis and related epithelial tissue: (2) 
Study of the lymphomatous disease. Mycosis 
fungoides. 



Epidermal Growth and Differentiation 

The aim of this program is to identify and 
characterize the varying and various biologic be- 
havior patterns of epidermis and related epithelial 
tissues under normal and pathological circum- 
stances, and to determine the natiire of influences 
that naturally exist, or that can be experimentally 
exerted, which predictably can alter or determine 
a specific pattern of response of the epithelial cell. 

It has been demonstrated over the past 40 years 
that embryonic epithelimn responds in markedly 
different patterns to environments of dift'erent con- 
nective tissue stroma. Thus, the normal eventual 
form and f miction of an epithelial cell is the result 
of the stromal environment in whicli it is found. 

Recent work in our Branch, involving auto- 
transplantation of epithelial cells into various 
anatomical sites in the human, has indicated the 
same is true for ijost-embryonic epithelia. 

It is readily apparent that the cutaneous epi- 
thelial cell possesses a wide potential range of 
speed of reduplication and pathways of differen- 
tiation, depending upon its anatomical location. 
For example, the rate of reduplication of epithelial 
cells of the hair root is known to be consideral)ly 
more rapid than those of the epidermis, altliough 
the precise rate of each has not heretofore been 
determined; and epithelial cells of the epidermis 
difl'erentiate and produce fibrous proteins which 



NATIONAL CANCER INSTITUTE 



43 



are different from those produced by the hair 
root cells. 

The direction of a portion of the investigations 
of this Branch to date has been to quantitate the 
rate of reduplication of the varioiis epithelial 
tissues of man and biochemically to define the 
products of differentiation. Tlii'ough these 
studies it has been found that the germinative cells 
of the hair root double their populations every 
twenty-four hours ; the time required for the epi- 
dermis to reduplicate itself has been found to be 
twenty-eight days, whereas the epidermis in the 
disease psoriasis requires but three days. Several 
insoluble proteins have been identified as products 
of the normal and abnormal epidermis and their 
peptide content studied. 

The studies of the epidermis in psoriasis have 
indicated that the lesion is one of benign epi- 
dermal hyperplasia (increased rate of reduplica- 
tion, increased mitotic index, excessive production 
of protein). The effects of mitotic "arrestors," 
antimetabolites, alkylating agents, and radiation 
on the lesion have been studied. These agents 
have been fomid to inhibit the rate of cellular re- 
duplication, but in the doses employed, showed no 
inhibition of differentiation (in this instance, 
keratinization) . On the contrary, keratinization 
is accelerated under these circumstances. 

A consistent pattern of mitotic activity has been 
elucidated in human hair roots. ]\Iitotic activity 
has been found to be con-elated with certain geo- 
metric data, regardless of the size of hair roots. 
Thus, the mitotic index of any given segment in 
the hair root can be formulated by the equation, 
X=113 — 0.58 Y, where Y is the distance (in 
microns) between the segment and the proximal 
tip of the connective tissue hair papilla. !N"o such 
relationship has been found for normal epidermis 
nor the hyperplastic epidermis of psoriasis, in both 
of which mitotic activity is confuied to the region 
of basal cells. The pattern of mitosis in basal 
cell tumors is being studied. 

In an attempt to detei-mme what factors pro- 
mote keratinization of the epidermal cell, split 
thickness specimens of skin have been cultured m 
vitro wider different conditions. Results of tlus 
woi'k suggest that a high pH of the culture 
medium and/or high CO2 tension of the ambient 
atmosphere promotes the keratinization process. 

Studies of basal cell carcinoma indicate that this 



neoplastic lesion can be characterized, hj an in- 
ability for its constituent epidennal cells to kera- 
tinize. Although chemotherapeutic drugs inhibit 
the growth of this lesion, no evidence has been 
f oimd that these compounds promote normal kera- 
tinization of the epidermal cell in psoriasis. In- 
hibition of growth basal cell tumors by metho- 
trexate, appears to be proportional to the mitotic 
activity of the tmnor. Attempts have been made 
to stimulate mitotic activity within tumors, with 
both ultraviolet light and X-ray, prior to admin- 
istration of methotrexate. Initial obsei-vations 
suggest that prior-irradiated tumors show a 
greater damage response from the drug than non- 
irradiated tumors. 

Mycosis Fungoides 

Approximately fifty patients with this rare 
lymphomatous disease have been studied and 
treated during the past eight yeai-s. Its histo- 
pathogenesis, from its onset in the skin to its in- 
volvement of internal organs, has been carefully 
investigated. Treatment with X-ray, high 
energy electrons, and several chemotherapeutic 
drugs has been given. Although remissions of 
the disease have been attained, no cm-ative I'e- 
sponses to these measures have been observed. 
Perhaps significant, however, is the fact that pro- 
longed remission has occurred in four of twelve 
patients in response to therapy with the drug 
Cytoxan, one patient remaining virtually free of 
lesions now for over one year in response to con- 
tinuous therap3'. 

A seemingly pertinent observation in this dis- 
ease has been the occurrence of a spontaneous 
"cure" in one patient following an allergic diiig 
eruption. Wliereas spontaneous remission of this 
disease has been unknown, this patient has been 
free of disease for over four j-ears. Transfusions 
of blood from this patient to another patient with 
the disease has been without effect. Attempts to 
provoke allergic skin eruptions in se'^-eral patients 
with the disease have been unsuccessful. 



ENDOCRINOLOGY BRANCH 

The studies of this Branch ha^-e been directed 
towards increasing our knowledge of the processes 



44 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



of growth differentiation and control of certain 
normal tissues and their neoplastic derivatives. 
These tissues include the endocrine glands and 
those organs whose maintenance or growth is de- 
pendent upon specific humoral agents. 

In the area of trophoblastic disease, follow-up 
studies of 30 patients who were successfully treated 
for metastatic disease revealed that relapse has 
not occurred after a remission of over one year. 
In methotrexate-resistant cases, Actinomycin D 
has proved a valuable therapeutic adjunct result- 
ing in a 50 pei-cent remission rate in this group. 
Methotrexate has been used in women with per- 
sistent evidence of disease following a hydatidi- 
form mole and has caused a complete remission 
in each of 8 cases. Since these patients are pro- 
spective victims of metastatic trophoblastic dis- 
ease, this response may be regarded as an example 
of specific cancer prophylaxis by chemical means. 
The endocrinological effects of trophoblastic dis- 
ease have been examined and a singular coinci- 
dence of hyperthyroidism noted. The hyperthy- 
roidism has subsided with treatment of the 
trophoblastic disease. 

Eight strains of human choriocarcinoma are 
being carried by serial transplantation in the 
hamster cheek-pouch. These strains are sensitive 
to many therapeutic agents some of which had 
been ineffective in the patient. The implications 
of this with respect to the usefulness of heter- 
ologous tumor transplants are being examined. 
Several classes of agents have been screened for 
therapeutic usefulness in this system and several 
alkaloids to Vincaleukoblastine proposed for 
clinical trial. 

Immunological techniques have been used in an 
attempt to distinguish between tumor gonado- 
tropin and normal gonadotropin. No differences 
were found notwithstanding the different dis- 
tribution of these hormones in plasma proteins. 

Among the humoral controls of tissue growth, 
the steroids play an important role. Methods 
have been devised for the analysis of several 
steroids whose precursors are important in adrogen 
synthesis. The precursors of several androgens in 
adrenal cancer have been investigated and were 
shown to arise via pathways not significant in the 
normal steroid sjoithesis. 

Adrenal androgen biosynthesis in gonadal 
dysgenesis was shown to be decreased suggesting 



that the genetic factors important in gonadal 
dysgenesis also influence the development of bio- 
synthetic pathways in the adrenal gland. 

The analj'sis of the prostatic response to ACTH 
has been continued and it was shown that thy- 
roxine and growth hormone enhanced the effect of 
ACTIi. Prolactin did not augment the effect of 
androgens on the growth of the ventral prostate. 

The pharmacological alterations of adrenal 
secretion by derivatives of compounds related the 
insecticide, DDT are being exammed in the dog in 
the hope of finding a less toxic agent than 
o,p' DDD. Complete chemical adrenalectomy in 
the dog could be obtained by prolonged admin- 
istration of o,p' DDD. 

Twenty-six patients with metastatic adrenal 
cancer have been treated with o,p' DDD. Hor- 
monal remission was achieved in 18 and tumor 
regression in 10. Comprehensive analysis of 
urinary steroids in these patients has uncovered 
defects of steroid synthesis in adrenal cancer and 
resulted in the finding that the increased excre- 
tion of tetrahydro substance S in patients with 
Cushing's syndrome is highly suggestive of 
cancer (Hertz and Lipsett) 

The pituitaiy hypothalamic level, a series of 
patients subjected to hypophyseal stalk section 
for metastatic breast of these hormones in plasma 
proteins. 

Among the humoral controls of tissue growth, 
the steroids play an important role. Metliods 
have been devised for the analysis cancer has been 
examined. The therapeutic effectiveness of stalk 
section was less than that of hypophysectomy. 
Persistence of the nonnal thyroid-pituitary rela- 
tionship can occur after stalk section. In the rat, 
it has been shown that the formation and release 
of thyrotroph! from the pituitary in vivo is greatly 
enhanced by hypothalamic tissue. The secretion 
of melanocyte stimulating hormone in the frog 
was demonstrated to be imder hypothalamic con- 
trol. Studies of the various factors affecting this 
melanocj^te response have been described to pro- 
vide a basis for the development of a bioassay 
method. These studies have been initiated to ob- 
tain some insight about the complex relationship 
between the central nervous system and the endo- 
crine system. 

Other growth factors are being examined. 
Growth hormone was ineffective in primordial 



NATIONAL CANCER INSTITUTE 



45 



dwarfism suggesting that this entity is due to an 
end-organ resistance. 

The adjustment of the normal subject to a 
variety of anabolic and catabolic agents has been 
determined using newly devised paired-tray tech- 
nique. This has the potential of answering sev- 
eral questions that camiot be handled by the 
classical balance teclinique. 



MEDICINE BRANCH 

Chemotherapy Service 

A number of new agents have been studied 
clinically as regards their ability to produce 
tiunor regression. These include Cytoxan, guanyl- 
hydrazone, vincaleukoblastine, leurocristine, di- 
chloromethotrexate, the terephthalanilides, and 
sarcolysin. A guanylhydrazone derivative, meth- 
yl-glyoxalbis-guanylhydrazone, has produced 
complete remissions in approximately 50 percent 
of adults vsdth acute myelogenous leukemia. This 
is tlie first major therapeautic advance to occur 
m this disease which previously was slightly re- 
sponsive to only one drug, 6-mercaptopurine. 
Guanylhydrazone has also produced tmnor regres- 
sions m patients with lymphosarcoma and mycosis 
fungoides. The toxicity of guanylhydrazone has 
been considerable and relates primarily to the 
gastrointestinal tract, the skin, and the bone mar- 
row. In collaboration with the Cancer Chemo- 
therapy l^ational Service Center and the Midwest 
Research Institute a number of related quanyl- 
hydrazone congeners are being prepared iu an 
effort to determine the active chemical sites, and 
hopefully to improve therapeutic index. Guanyl- 
hydrazone C^* has been synthesized and initial 
pharmacology studies in rodents are under way. 
The mechanism of action of guanylhydrazone is 
independent of that for the other known cancer 
Chemother apeutic agents in that cross resistence 
does not occur. 

Tlie periwinlvle alkaloid, vincristin, has also 
been shown to be capable of inducuag remissions 
in children with acute leukemia and tmnor regres- 
sion in a large proportion of patients with Hodg- 
kia's disease and lymphosarcoma. There is pre- 
liminary evidence that tliis compound is active in 
patients with carcinoma of the breast and per- 



haps certain other patients with solid tmnors. 
Dose schedule studies with this compound in man 
reveal that the tolerated dose per imit time is inde- 
pendent of the schedule. '^"\1iile vinblastine, a 
related alkaloid, also produces tumor regression 
in patients with Hodgkin's Disease and lympho- 
sarcoma, it was mactive in patients with acute 
leukemia. Thus, major differences not only in. 
toxicity but in the therapeutic effects exist between 
these chemically very closely related Periwinkle 
alkaloids. (Carbone and Karon) Vincristin pro- 
duces marked metaphase arrest. In the bone mar- 
row the proportion of cells in metaphase rises from 
a control of 10/1000 to in excess of 70/1000 at 12 
to 24 hours after intravenous administration. 

A comparative study of Cytoxan, uracil mustard 
and nitrogen mustard in patients with various 
solid tumors including lymphoma has revealed no 
significant difference in either the antitumor ac- 
tivity of these agents or in the overall toxicity. 
Sarcolysin, a phenyalanine mustard, is the first 
drug in our hands to produce significant antitumor 
effects in patients with multiple myeloma. Though 
complete regressions have not occurred, substantial 
decrease in abnormal protein production, a de- 
crease in myeloma cells of the marrow, bone heal- 
ing, and improvement in anemia have occurred in 
a substantial inumber of patients. A comparative 
study of urethane versus placebo in patients with 
multiple myeloma has revealed that urethane is in- 
active and that spontaneous improvement of any 
sort is rare in patients with multiple myeloma. 
Other clinical studies include comparative dose- 
response studies of 6-mercaptopurine and 6- 
mercaptopurine riboside in patients with solid 
tmnors; a comparative study of fluorouracil, 
fluorodeoxy uridine and methotrexate in patients 
with carcinoma of the breast and colon ; studies of 
azauridine, both in terms of its clinical anti- 
leukemic effect and its ability to inhibit the enzyme 
orotidylic decarboxylase in patients with chronic 
myelogenous leulvemia; and studies of tlie effects 
of Cytoxan, fluorom-acil, vmblastine and duazo- 
mycin A in patients with carcinoid S}mdrome. 
This latter study was undertaken when it was ob- 
served that certain of these compounds are highly 
active in a serotonin producing mast cell rodent 
tumor. In association with timior regi-ession 
qualitative and marked quantitative changes in 
tryptophane metabolism occurred. 



46 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



In association with tliese clinical trials there is 
contimied experimentation with and improvement 
of experimental design as well as extension of our 
knowledge of the natural history of advanced neo- 
plasms in man. The results of these quantitative 
antitumor studies have been carefully compared to 
a nmnber of the screens, particularly the rodent 
tumor screen, used in selecting antitumor agents 
for tumor trial. In this regard the L1210 mouse 
leukemia system which in general would appear 
to predict fairly well for human acute leukemia 
failed to disclose significant activity for vincristin, 
a compound which was found to have considerable 
activity in human acute leukemia. The rat 
lymphomas and leukemias which had been exten- 
sively used in the evaluation of alkalating agents 
would appear to correlate poorly with human 
tumors in terms of ranking therapeutic agents. 
(Frei and Rail) 

Tlie use of plasmapheresis to obtain platelets 
from normal donors for the prevention and con- 
trol of bleeding in thrombocytopenic recipients 
has been attended with considerable success. 
Eighty-five pei-cent of recipients will have a rise 
in their platelet count and will not demonstrate 
diminishing response with successive transfusions. 
In addition to becoming an established thera- 
peutic procedure it allows for the application of 
in vitro tecloniques for measuring platelet function 
and their collation with the in vivo observations 
and the study of the efficacy of the various 
methods of platelet preservation. More recently 
the same general techniques have been applied in 
the acquisition of white cells from donors with 
chronic myelogenous leukemia. One hmidred 
transfusions of such cells have been given to 
granulocytopenic individuals, mainly patients 
with acute leukemia. A significant rise in granu- 
locytes in the recipcient occurs in the majority of 
patients. Many of tlie patients had severe infec- 
tions at the time of transfusion, for example, 
pseudomonas septicemia. In the majority of 
these, prompt control of the infections was 
achieved. The implications of the observation 
that we can now control both hemorrhage and in- 
fection in patients with acute leukemia are major. 
From 90 percent of the patients with acute leuke- 
mia and in excess of 50 percent of patients with 
malignant disease generally die of these complica- 
tions. 



The technique for chromosome stud}- of clinical 
material has been simplified and applied in 40 
patients with acute leukemia and 10 patients with 
chronic leukemia, 4 patients with multiple mye- 
loma and a number of other disease entities whicli 
allow for the acquisition of relative homogenous 
cellular preparations. Abnormalities both in 
number and quality of chromosomes are relatively 
frequent but as yet, with the exception of the 
Philadelphia chromosome in chronic myelogenous 
leukemia, no consistent patteni has evolved. De- 
tailed clinical correlative studies are under way to 
look for relationships to the chromosome abnor- 
malities. 



Clinical Pharmacology and Experimental Thera- 
peutics Service 

Major emphasis continues to be given to the 
problem of drug distribution. Considerable 
progress has been made concerning our under- 
standing of factors which influence the entiy and 
exit of diiigs in the central nervous sj'stem. Exit 
from the central nervous system may result from : 

1. Passive diffusion for lipid soluble compounds. 

2. Bulk flow. This obtains for large lipid in- 
soluble compounds. Evidence concerning the im- 
portance of this latter mechanism was obtained 
when it was observed that Acetazolamide, a com- 
pound which decreases cerebral-spmal fluid pro- 
duction, and therefore bulk flow, will decrease 
elimination of large lipid insoluble compomids. 

3. Sterospecific active transport. In vitro 
studies of the animal choroid plexus have de- 
lineated this mechanism. Two related problems 
are being pursued. One concerns the absolute 
rate of production of cerebi-al-spinal fluid and the 
other concerns dynamic studies of tlie entry and 
egress of magnesium from the cerebral-spinal 
fluid. This latter study was prompted by the ob- 
servation that the concentration of magnesium in 
the cerebral-spinal fluid is greater than that in tlie 
blood. The broad implications of these observa- 
tions for a drug development program are ap- 
parent. Clinical application of these concepts 
and techniques have been made, particularly as 
they may be applied in the treatment of that im- 
portant clinical entity, meningeal leukemia. Tlie 
tolerated dose and the magnitude of entry and 
egress of methotrexate from the central nen'ous 



NATIONAL CANCER INSTITUTE 



47 



system has been studied. Since methotrexcate does 
not pass the blood brain barrier in any quantity it 
must be delivered by lumbar puncture. The dif- 
fusion throughout the subarachnoid space from 
this site has been studied in monkeys usmg I"^ 
labeled rose bengal. Certam mechanical maneu- 
vers at the time of lumbar tap may assure adequate 
distribution. 

The Medicine Branch has a major commitment 
to the folic acid antagonist area which extends 
from synthesis up through clinical trial. Studies 
with radioactive chlorine labeled dichlorometho- 
trexate (DCM) have revealed a considerable var- 
iation in organ distribution depending upon the 
specie. By the use of ion exchange column tech- 
niques and UV spectroanaylses the metabolites of 
methotrexate and dichloromethotrexate have been 
defined. In contrast to methotrexate, dichlorome- 
thotrexate has a major metabolite, 4,7-dihydroxy 
folic acid. The magnitude of convereion to this 
metabolite also varies from specie to specie and 
this probably is the major explanation for the les- 
ser toxicity of DCM. 

The mechanism of resistance to thiopurines in 
tissue culture and in many animal tumor systems 
relates to the selection of cells incapable of con- 
verting 6-MP to the active nucleotide. These 
studies have been extended to man and preliminary 
evidence would suggest that deletion of the enzyme 
involved does not occur in human leukemia cells 
resistant to 6-mercaptopurine. 

Studies of carcinogenic materials in newborn 
animals including newborn monkeys have indi- 
cated that squamous cell papillomas can be pro- 
duced regularly when methylcholanthrene in olive 
oil is injected intradermally. In addition to car- 
cinogenesis, the reproducibility of this system is 
such that it may be used in chemotherapy studies. 



LABORATORY OF PATHOLOGY 

The Laboratory of Pathology has a more com- 
plex organization than many other laboratories 
of the National Cancer Institute. This is because 
the Pathologic Anatomy Branch performs fvuic- 
tions related to patient care and clinical research 
in the Clinical Center; the Histopathology Lab- 
oratory prepares microscopic sections on a service 
basis for the entire Institute ; and another group 



in the Cancer Induction and Pathogenesis Section 
is devoted primarily to non-clinical research. 
There is a close association among all the patholo- 
gists in the Laboratory of Pathology and frequent 
collaboration. Opportimity is given to members 
of the Pathologic Anatomy Branch to do research, 
and pathologists not directly responsible for the 
autopsy or surgical biopsy service may still observe 
autopsies and avail themselves of hmnan material. 
The work in this laboratory is not restricted to 
a single project, or to a group of closely related 
projects but each pathologist follows his particu- 
lar line of interest and training. It is therefore 
convenient to divide this summary into a number 
of sections. 

Collaborative Research 

It is recognized that many research projects at 
the National Cancer Institute require the collabo- 
ration of a pathologist, especially in the final 
evaluation of the effect of an experimental proce- 
dure on laboratory animals. The Laboratory of 
Pathology has always tried to make this assist- 
ance available. The pathologist may take an 
active part in plamiing an experiment and in fol- 
lowing it through; he may take the responsibility 
for all autopsies and histologic diagnoses in an ex- 
periment; he may review only the histologic sec- 
tions in a given experiment; or he may seiwe as a 
consultant to review selected material with no re- 
sponsibility for the entire experiment or its pub- 
lication. Finally, he may make use of material 
accmnulated by other investigators for independ- 
ent studies concerning pathologic alterations. It 
is emphasized that full collaboration of the pathol- 
ogist at the time the experiment is planned is the 
most satisfactory arrangement for it insures the 
best and most economical selection of material for 
pathologic studies. Examples of collaborative re- 
search now in progress are: a) Dr. Stewart and 
Dr. Snell with Dr. Morris of the Laboratoi-y of 
Biochemistry in studies of the effect of new carci- 
nogenic agents in rats. As a result of these studies 
a monograph on lesions produced by N,N'-2, 7- 
fluorenylenebisacetamide has been published, b) 
Dr. MacCardle with Dr. Potter of the Laboratory 
of Biology on the cytology of induced plasma cell 
tmnors. These studies have disclosed a cytologic 
correlation between the neoplastic cells in partic- 



48 



AlOniAL REVIEW OF INTRAMURAL RESEARCH 



ular transplantation lines, and indicate that 
different globulins are produced in the protein- 
secreting plasma-cell tumors at different stages of 
maturation of the plasma cell, c) In observations 
made by Dr. MacCardle with Dr. Frederic Bartter, 
a new clinical syndrome has been f oimd in which 
hyperplasia of the juxta-glomerular apparatus ac- 
companies hyperaldosteronism and hyperkalemia 
with a normal blood pressure, d ) Dr. O'Gara with 
Dr. Kelly of the Medicine Branch, NCI, on the ef- 
fects of mjecting chemical carcinogenic agents into 
newborn animals. Tliis work revealed that ex- 
tremely small doses were effective, e) Dr. Dunn 
with Dr. Moloney of the Laboratoiy of Viral 
Oncology in a study of morphologic changes pro- 
duced by his virus. This study showed that a num- 
ber of changes preceded the development of 
lymphocytic leukemia, f )Dr. Dunn with Dr. An- 
dervont, Laboratory of Biology, in a review of 
tumors appearing in wild mice. In tliis study, the 
pathologist did no more than make a diagnosis of 
the tumors, but the results showed that morpho- 
logically these tumors closely resembled tumors 
observed in inbred mice, g) Dr. Swarm with Dr. 
Morris and Dr. Dyer on carcinogenesis in rats 
with congenital hyperbilirubinemia. Also with 
other members of the NIH staff where his special- 
ized knowledge of radioisotopes and autoradio- 
graphic techniques was required, h) Dr. Banfield 
in a nmnber of mvestigations where electron 
microscopy was required. Examples are studies of 
Whipple's disease, psoriasis, mouse thymic agent, 
acanthosis nigricans, and the Arthus reaction, i) 
Dr. Mahngren with other members of the NIH 
staff where the flourescent antibody technique is re- 
quired, j ) Dr. Sidransky with Dr. Morris on the 
pathologic changes produced by N-2-fluorenly- 
acetamide in male and female rats. 



Laboratory animals 

Tliis is a continuing activity in the Laboratory 
of Pathology. Precise knowledge regarding the 
normal anatomy of laboratory animals is often 
lacking, particularly as regards variations in in- 
bred strains and the alterations appearing when 
animals reach the age when cancer can be expected. 
The investigator using laboratory animals must 
know his basic material for he relies upon it much 
as a chemist relies upon the substrates in a reaction. 



Dr. Stewart and Dr. Snell are accumulating data 
on aged rats from five inbred strains. For the 
second year, Dr. Snell has given a lecture as part 
of a course at the Armed Forces Institute of Pa- 
thology on the use of Laboratory Animals in Ke- 
search. Tliis lecture describes spontaneous lesions 
in old rats and the incidence of cancer in the vari- 
ous strains. Dr. Snell is also collecting data on the 
Mastomys, a species of rodent recentlj"^ introduced 
to the Laboratory. Dr. Banfield, Dr. Dunham, Dr. 
Herrold, Dr. Chu, and Dr. Swarm are collecting 
data on the hamster, a species now popular in can- 
cer research, and about wliicli our knowledge is 
still limited. Dr. Dimn continues to collect in- 
formation on the endocrine system of the mouse. 
Dr. Swarm has mider observation a breed of rats 
with an inborn error in tlie matabolism of biliru- 
bin. Basic data are required before many of these 
animals can be used with greatest profit. 

Transplantable Tumors 

The first studies on transplantable tumors in 
animals were made by pathologists and this in- 
terest has continued. The interpretation of the 
effects of certain tumors on the host has given 
important information regarding tlie function of 
normal organs, especially the endocrine organs. 
Work on transplantable adrenal cortical carcino- 
mas is being continued by Dr. Snell and by Dr. 
Mulay, each of whom is carrying a tumor of this 
type. Dr. Mulay has found a difference in the 
secretion of the tumor and material obtained from 
the normal adrenal gland. Dr. Mulay has also 
found that hepatogenesis is modified by the hor- 
monal state of the rat and that changes in adreno- 
cortical chemistry precede neoplastic changes. 

Dr. Banfield, assisted by ISIrs. Darlene Brindley, 
is studying a transplantable reticulum cell sar- 
coma in hamsters which is transmitted from one 
animal to another in the same cage. It is the first 
instance of such natural transmission that has 
been found recorded. The possibility that a cell 
transfer and implantation occurs when other 
hamsters eat the cancerous tissue is being investi- 
gated, and also the possibility of infection by a 
transmissible agent is being explored. A second 
tumor of this type has now been found. Dr. 
Swarm is continuing his studies of a transplant- 
able teratoma of the testis. This tumor produces 



NATIONAL CANCER INSTITUTE 



49 



cartilage, and the behavior of the neoplastic car- 
tilage, and cartilage from normal animals after 
subcutaneous transplantation is being compared. 

Virus Research 

Members of the Laboratoiy of Pathology have 
been interested in exploring the morphologic 
alterations produced by oncogenic viruses both 
in tissue culture and in the experimental ani- 
mal. Dr. Clyde Dawe is carrying on fundamental 
studies in observing the alterations produced by 
the polyoma virus on whole organ tissue cultures 
of salivary glands of mice. It is disclosed for the 
first time that the action of the mesenchyme pro- 
foundly affects the reaction of epithelial tissue to 
the virus. These studies also prove that tissue 
from the adult is as susceptible as tissue obtained 
from the newborn. This finding is contrary to 
in vivo observations for the effect of the virus 
has been largely restricted to newborn animals. 
Dr. Dawe has also shown that if adult salivary 
gland tissue is exposed to the virus for two hours 
and then transferred subcutaneously in a mouse, 
a salivary gland type of tumor will develop at 
the site of implantation. This observation opens 
up many possibilities for future research and con- 
tinuing work with this system offers a hope that 
the mechanism by wliich the virus produces its 
oncogenic effect may yet be disclosed. 

Dr. Eabson has found that a strain of polyoma 
virus which lost its oncogenic property when 
grown in cells in a milk medium regained its onco- 
genic potency when grown in serum. This find- 
ing has fundamental significance for it proves that 
an alteration may be induced in the virus as well 
as in the host. The milk-medium strain with a 
low oncogenicity forms a small plaque when com- 
pared with the strain having greater oncogenic 
potency. Now that the actuality of oncogenic 
viruses is generally accepted it becomes urgent that 
the mechanisms by which they operate be deter- 
mined. The oncogenic outcome is the result of 
an interaction between the virus and the host, and 
both elements of this interaction require intensive 
study. Other observations made by Dr. Eabson 
show that the polyoma effect first appears in the 
nucleus and terminally in the cytoplasm. The ef- 
fect of polyoma virus in Mastomys is being invest- 
igated for it is known that the effects of this virus 



vary in different inbred strains of mice and in dif- 
ferent species of animals. If viruses play a part 
in human cancer, in order to prove this, we must 
understand the variable manifestations which they 
produce in non-human species. Dr. Eabson's at- 
temps to recover a virus from human cancer have 
so far been masuccessful. 

Dr. Dumi has worked in collaboration with Dr. 
Moloney, Dr. Eauscher, Dr. Manaker, and Dr. 
Bryan of the Laboratory of "Viral Oncology in 
pathologic studies of animals infected with leu- 
kemia viruses and with an agent obtained from a 
human gastric cancer. With the Moloney virus 
the most interesting finding is that in intact 
BALB/c mice and rats, the first neoplastic lympho- 
cytic cells are found in the thymus, but this find- 
ing is preceded by hyperplasia in the blood-form- 
ing organs and atrophy of the thymus. When the 
Moloney virus is given to thymectomized mice, the 
incidence of lymphocytic neoplasms is reduced 
almost to zero, but occasional cases of granulocytic 
leukemia (extremely rare in BADB/c mice) and 
frequent cases of reticuloendothelial dysplasia, 
often resembling Hodgkin's disease in man, ap- 
pear. This is of considerable interest in the con- 
troversy regarding the relationship among dif- 
ferent types of malignant lymphomas in man. 
The findings in mice suggest that the same stimu- 
lus may produce a lymphocytic, a granulocytic, or 
a reticulum cell neoplasm, depending upon the 
reaction of the host. It is not necessary to postu- 
late a separate virus for each of these diseases. 
The action of the Moloney virus also shows that a 
derangement of the reticular tissues precedes the 
appearance of the neoplasm, and the action of the 
-idrus is indirect. A virus also obtained hj Dr. 
Moloney from an imdifferentiated plasma cell 
neoplasm and the leukemia virus obtained by Dr. 
Manaker seem to affect the host in much the same 
way as the first Moloney Anrus, but more work on 
these is required. Pathologic studies on mice with 
the Eauscher virus are only at the beginning, but 
it api^ears that a lymphocytic neoplasm begins in 
the thymus, as with the Moloney virus. The neo- 
plastic condition, however, is preceded by an ex- 
treme erythrocytopoiesis, with enormous enlarge- 
ment of the spleen which frequently leads to 
hemorrhage and death before the neoplasm de- 
velops. With the Moloney and the Eauscher 
virus we appear to have two distinct agents each 



50 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



leading to the same form of lymphocytic neo- 
plasm, but preceded by very different conditions 
in the host. The agent recovered by Dr. Kay 
Bryan from the hmnan gastric cancer has pro- 
duced a significant number of renal tumors (a very 
rare condition) in BALB/c mice. These renal 
tumors are always accompanied by cysts in the 
liver and occasionally by carcinomas of the acinar 
cells of the pancreas of a type never before seen 
in mice. The evocation of rare and previously 
unknown lesions in the mouse, and the association 
of three apparently unrelated conditions has some 
similarity to the action of the polyoma virus. 

Dr. Eobert Love, before leavmg the National 
Cancer Institute, was engaged in cytochemical 
studies of the nucleic acids in normal and neo- 
plastic cells infected with riboviruses and deoxy- 
riboviruses. Using his toluidine blue molybdate 
staining procedure he has shown that the earliest 
alteration in P388Di cells infected with polyoma 
virus is enlargement of the nucleolinus which is 
subsequently extruded and then forms numerous 
particles in the nucleoplasm. 

Chemical Carcinogenesis 

This continues to be a fruitful field for study by 
the pathologist, since pathogenesis is always of 
major interest. A notable achievement was work 
performed by Dr. Mearl Stanton which provides 
(1) a new method for the induction in rats of lung- 
tumors that histologically resembled human lung 
tumors, and (2) the demonstration that two factors 
acting concurrently will induce lung tumors in 
rats, while neither was effective alone. Multiple 
infarcts were produced in rat Imigs by the intra- 
venous injection of a fluorocarbon, and when this 
treatment was combined with mjections of methyl- 
cholanthrene, bronchiogenic tumors resulted. This 
observation is higlily significant for human lung 
cancer because exposure to a carcinogen may be 
relatively harmless in a healthy lung and dan- 
gerous in a diseased lung. 

Several carcinogenic studies have been stimu- 
lated by observations on the geographic distribu- 
tion of cancer m man and on studies of special 
types of human cancer. Dr. O'Gara is trying to 
induce cancer by exposing animals to material ob- 
tained from utensils used by African natives with 
esophageal cancer. Doctors Herrold and Chu are 



studymg the development of uterine cervical can- 
cer in mice and hamsters. Dr. Dunham is devising 
techniques to compare the possible carcinogenic 
effect of adsorbates obtained from the drinking 
water of a city with a high bladder cancer rate 
(New Orleans) with water from a city with a low 
rate. Dr. Herrold and Dr. Dmihani have pro- 
duced kmg cancer in Syrian hamsters by the intra- 
tracheal instillation of benzo (a) pyrene. Thorium 
wire and urethane in the bronchus were ineffective. 
They have also tested the hamster cheek pouch for 
carcinogenic response to different substances, and 
found this site generally unresponsive. Dr. 
Swarm continues to study the late effects of the 
injection of thorimn dioxide in man and in mice, 
in rats and in rabbits. Similar cancers of the 
liver are developed in all four species. 



Geographic Pathology 

The project comparing uterine cancer in various 
ethnic groups in New York City, Israel, and Wash- 
ington, D.C., has been completed and a manuscript 
is in preparation. A similar survey of cancer of 
the uterus m Negro women in New York City and 
in Washington, D.C., is being analyzed. A project 
is in progress to survey bladder cancer in New 
Orleans and to look for possible factors to accoimt 
for the frequency in that city. Dr. Herrold is 
continuing her collection and review of lung cancer 
among Veterans. AU cases are examined his- 
tologically and Kreyberg's classification has 
f)roved satisfactory. Any relationship of smoking 
to the histologic type will be considered when the 
findings are tabulated. Dr. O'Gara visited the 
Transkei Region of South Africa where a high 
incidence of cancer of the esophagus is reported 
among the natives. Since his return he is testing 
various possibilities on laboratory animals for 
etiologic factors in human esophageal cancer. 



Plasma Cell Neoplasms in Mice 

Dr. Malmgren has employed the fluorescent anti- 
body technique to demonstrate that antisera to 
globulins in plasmacytomas is localized within the 
tumor cells. Dr. MacCardle has determined that 
a corelation exists between the cytology of the 
neoplastic plasma cell and the type of protein it 



NATIONAL CANCER INSTITUTEi 



51 



produces. Dr. Kobayashi found that all trans- 
plantable plasma cell neoplasms that he studied 
would produce osteolytic lesions if introduced in- 
travenously. Osteolysis therefore appears to be 
a special property of the neoplastic plasma cell 
and is not restricted to only a few transplant lines. 
Kidney lesions, however, are restricted to those 
transplant lines in BALB/c mice in which a 
Bence- Jones protein has been demonstrated. 

PATHOLOGIC ANATOMY BRANCH 

In addition to duties connected with the surgical 
biopsy, exfoliative cytology and autopsy service of 
the Clinical Center, the Pathologic Anatomy 
Branch pursues a number of research studies re- 
lating to hmnan pathology. These are described 
in detail in the reports from various members of 
the Pathologic Anatomy Branch. Publication of 
the pathologic studies is often incorporated in 
studies by clinicians in which the biopsy or au- 
topsy findings add significant iaformation. The 
pathologist may also make case reports where the 
findings are primarily of pathologic interest. In 
addition to this, several studies have been carried 
out where the autopsies of nmnerous cases are re- 
viewed and the information is synthesized in order 
to afford a unified concept. An example of this 
is a study by Dr. Louis B. Thomas on the skeletal 
and nervous system lesions in acute leukemia. 
This human study is now being correlated with a 
study of the central nervous system lesions in mice 
given the L-1210 lukemia and treated with Cy- 
toxan. In a similar fashion the Cytology Section 
examines material from patients for diagnostic 
purposes, and is also engaged in researcli acti^d- 
ties related to technical improvements of methods 
to recover cancer cells from the blood and other 
fluids and also in estimating the prognostic sig- 
nificance of circulating cancer cells. Dr. Chu by 
employing her skill in the cytodiagnosis of human 
vaginal smears has followed the painted uterine 
cervices of hamsters with a carcinogen. Carci- 
noma resulted and a comparison of the histologic 
appearance and the cells in vaginal smears showed 
that a good correlation existed. 

Dr. John H. Edgcomb has been an exchange 
fellow in Moscow since June 23, 1961. He expects 
to return at the end of December but will soon 
go to Ghana to head a research laboratory to be 



established there. While iu Moscow he has studied 
the histochemistry of transplantable melanomas 
and participated generally in the activities of the 
Institute of Experimental and Clinical Pathology. 
His reports of his life in Russia and observations 
of cancer research have been valuable and inter- 
esting. 

Dr. Thomas has investigated the effects of schis- 
tosoma from Egypt and from the Gold Coast on 
the bladder epithelium of hamsters. No bladder 
cancers have been found. This negative evidence 
is of interest in considering the problem of the 
high bladder cancer rate in Egypt and the fre- 
quency of schistosomiasis. 



LABORATORY OF PHYSIOLOGY 

Because of the diversified training and endeavors 
of this laboratory, the annual report presents a 
number of avenues of research. Yet, in spite of 
tliis, the ultimate goal sought is to throw light on 
the cancer problem. In general, all the research 
is directed towards a study of tumor-host relation- 
ship m vivo. Wlierever supplementary data or 
exploratory data from in vitro experiments can 
assist the studies in vivo, such experiments are 
encouraged. 

Dr. Pratt is oriented toward the physiological 
and biochemical systems that exist in the tumor- 
bearing animals. The large number of param- 
eters resulting in such a study made data proc- 
essing and computer utilization a necessary ad- 
junct. As a result, a digital computer (IBM- 
1620) was obtained. The instrument has two 
main uses: (1) to act as a data processor for 
monitoring and directing long-term experiments, 
and (2) to afford a powerful computational facil- 
ity for developing an applied mathematical meth- 
odology for research projects. He has been (a) 
formulating mathematical models for the inter- 
pretation and evaluation of cancer chemotherapy 
screening data, (b) with Dr. Thomas of the Path- 
ological Anatomy Branch, he is setting up a sys- 
tem for storing and retrieving information of 
pathology diagnosis, (c) witli Dr. Sober, he is 
developing chemical, mathematical and com- 
puter programming techniques to describe the 
arrangement of mononucleotides within jjolynu- 
cleotides, and (d) continuing his study on the 



643351—62- 



52 



AISTNTLTAI, EEVIEW OF ESTTRAMURAL RESEARCH 



total metabolism (energy, carbon, nitrogen and 
water) in normal and tumor-bearing rates. 

Dr. Shack is continuing his elegant techniques 
in attempts to characterize and compare nucleic 
acids and related compounds, as well as enzjanes 
and enzyme inhibitors of normal and malignant 
tissues. He has been (a) making intensive studies 
on the action of bivalent ions (Ca, Mg, Sr) in 
the activation of deoxyribonuclease, and (b) de- 
veloping methods wliich appear to offer promise 
of separating nuclei acids into their component 
fractions for further identification. He has dem- 
onstrated that although Ba, Ca and Mg are poor 
activators of ribonuclease by themselves, they 
seem to increase the activity of Mg ions when 
added to the latter. Tliis strongly suggests that 
two metal ions are concerned with the formation 
of a nucleic acid-protein complex. As a result, 
this system may be a useful model to study the 
factors involved in the formation and stabiliza- 
tion of other types of nucleoproteins. One of the 
major problems involved in characterizing nucleic 
acids is accounting for the partial denaturation 
whicli occurs during isolation. By employing 
electrophoretic techniques, Dr. Shack has been 
able to demonstrate the extent of the denaturation 
and which appears to be related to the proportions 
of the various purine and pyrimidine bases. It 
now makes it possible to separate nucleic acids of 
different composition with exact information on 
controlled denaturation. Separation of nucleic 
acid of different composition following controlled 
denaturation is now possible. 

Dr. Eabinovitz continues his studies on action 
of inhibitors which produce a "biochemical lesion" 
in protein synthesis at the protein assembly site, 
namely the ribosome. In studying the effect of the 
antibotic, puromycin, on tlie Ehrlich ascites tumor 
cell, he has produced what he calls a dissociative 
lesion where the partially assembled proteins are 
prematurely released from the ribosome. Employ- 
ing the lysine analog, S-(^-aminoethyl) cysteine 
in the rabbit reticulocyte, there occurs what he 
calls an accimiulative lesion whereby the assembled 
proteins either cannot be released as a final prod- 
uct or can be released with difficulty and therefore 
appear as excess precursors on the ribosome. 
These results suggest there may be a qualitative 
difference in the behavior of ribosomes of mam- 
malian cells during protein synthesis. 



Dr. Winitz is continuing his studies with his 
water-soluble diet. He is attempting to determine 
the minimal protein requirement in terms of amino 
acid intake. It has been suggested that the free 
amino acid levels in the blood of a given animal 
species in the fasting state might serve as a precise 
indicator of the dietary essential amino acid re- 
quirements of that species. Hence, time-consum- 
ing and expensive nitrogen balance studies could 
be eliminated. Indeed, Dr. Winitz has demon- 
strated that diets prepared according to the pro- 
portions of the amino acids in the plasma in- 
duced growth comparable to the best available 
known synthetic diet and compared favorably 
with Purina chow. Studies to elucidate the im- 
plications of this diet in studying nutritional de- 
ficiencies and the impact of a growing tumor on 
tlie host are imderway. 

Dr. Wollman is continuing his studies on the 
early events leading to the formation of thyroid 
hormone in normal thyroid tissue. He has made 
extensive studies on the iodide-concentrating 
mechanism and the site of formation (including 
kinetics of formation) of protein-bound iodine. 
More recently he has turned his attention to ex- 
amining the mechanism of the secretion of thyroid 
hormone. In collaboration with Dr. S. S. Spicer, 
he has identified intracellular colloid droplets in 
thyroid which have the properties of lysosomes. 
These droplets contain colloid derived from the 
lumen of the follicle as well as hydrolytic enzymes. 
It appears therefore that the droplet is an intra- 
cellular organelle in which thyroxine is released 
from peptide linkage in thyroglobulin prior to 
secretion of the thyroxine into the blood. Dr. 
Wollman has also developed a variety of func- 
tional and non-fimctional thyroid tumors in the 
Fischer rat. It was not possible to distingiiish be- 
tween the functional and non-functional follicles 
on the basis of shape, size or staining properties. 
Some of the functional tumors can compete effec- 
tively with the thyroid for a hormone precursor. 
He was able to divide these tumors into two 
classes: (a) tumors which suppressed severely the 
ability of the thyroid to clear radioiodine from 
the blood, and (b) tumors which had almost no 
effect on thyroid clearance. 

Dr. Elkind continues liis elegant research on the 
kinetics of the growth and survival properties 
of surviving cells (as a result of exposure to vari- 



NATIONAL CANCEE INSTITXJTE 



53 



ous doses of X-irradiation) after the termination 
of the di^asion delay period. He has demonstrated 
that the post-delay growth is not synclironous (as 
commonly presmned) but exponential with a dou- 
bling time very close to that of unirradiated cells. 
Thus the so-called "mitotic block," presimied to be 
the explanation of the irradiation-induced delay, 
is not a true block but rather a delay. Dr. Elkind 
is attempting to formulate a mathematical model 
of the growth and radiation responses of cells and 
tissues. If successful, this model should contrib- 
ute much to our understanding of radiation ef- 
fects. He plans to study changes in nucleic acids 
and proteins in cells after irradiation. 

Dr. Reid is continuing his study on urinary ex- 
cretion patterns in human leukemia with special 
reference to nucleic acid congeners. During tlie 
year, Dr. Eeid had a total of 13 patients under 
investigation : 3 normals on a free diet and on the 
control diet used for pathology studies ; 3 mitreated 
cases of acute myeloblastic leukemia ; i untreated 
cases of acute lymphoblastic leukemia; and 3 of 
the acute lymphoblastic cases in remission after 
therapy. Because of the wide variety and number 
of components present, a good deal of time has 
been spent in the reduction and analysis of the pro- 
file data and upon the systematic investigation of 
the composition of the mixed peaks obtained in 
the chromatographic procedure. The introduc- 
tion of data processing machines into the Labora- 
tory has been higlily successful and information 
of a concrete nature should be available very soon. 
Preliminary work has unequivocally demonstrated 
the presence of pseudouridine. Its excretion has 
been shown to be diet dependent. There is no rela- 
tion between pseudouridine excretion and my- 
eloblastic leukemia. There is a definite relation- 
ship between pseudourdine excretion and lympho- 
blastic leukemia. 

Dr. Millar and Dr. White liave continued to 
study tumor-host relationships using the Walker 
256 tumor. We have demonstrated clearly the 
great demand for ISTa ions by tumor-bearing ani- 
mals (far in excess of that supplied in tlie normal 
diet). In collaboration with Dr. J. O. Davis 
(Laboratory of Kidney and Electrolyte Metabo- 
lism, NHI), a study was made of the corticoste- 
roid output of the adrenal gland. Aldosterone 
output was found to be markedly increased over 
normal. 



These investigators have also made some pre- 
liminary studies of the isolated protein of Walker 
tumor as a source of nitrogen for growth. Two 
tentative (need verification) obsei-vations have 
been made: (a) animals ingesting a 6 per cent 
casein diet show a 20 per cent higher increment 
in growth when supplemented daily with 300 mg. 
of tumor protein than with 300 mg. of casein, sug- 
gestmg a higher growth efficiency of tumor pro- 
tein, and (b) animals bearing tumors and ingest- 
ing tumor protein (20%) grow statistically larger 
and have larger tumors than do a similar group 
of animals ingesting casein protein (20%). 

In addition, Dr. Millar in collaboration with 
Dr. Winitz and Dr. Pratt are studying the gross 
body composition in normal and tumor-bearing 
rats on a completely water-soluble, chemically- 
defined diet. It is now possible to compartmental- 
ize each component that contributes to body 
growth and ultimately demonstrate the impact 
of the tumor on the host in terms of nitrogen, 
water, and electrolj^es. 

Dr. Willie Smith has been studying spontaneous 
and induced recovery from radiation damage. 
She finds that age is an important factor in spon- 
taneous recovery and that its effect may vary with 
particular components of recovery, thus gi-anu- 
locjiie recovery and recovery from gastrointestinal 
damage seem to improve with increasing age of 
the mouse while recovery of the immune mecha- 
nism occurs somewhat more rapidly in young ani- 
mals. A single injection of endotoxin before or 
immediately after irradiation leads to an early 
recovery of bone marrow cellularity as well as 
peripheral granulocyte, erythrocyte and platelet 
counts. There is also an increased survival. 
Toxic manifestations of endotoxin are undesirable 
and other products which may be less toxic are 
being sought. Studies of a similar nature are be- 
ing conducted with colchicine and colchicine 
derivatives. 

Dr. Draper is studying i-adiation immimology 
to contribute to our imderstanding of both radia- 
tion effects and the nature of the immune process. 
He has demonstrated the reduction of the dele- 
terious effects of a given dose of radiation bj' frac- 
tionation (over-all exposure time is increased and 
the average dose thereby lowered) and has ex- 
tended this observation by using hemolysin for- 
mation in rabbits as an indicator system. These 



54 



ANNUAL REVIEW OF ESTTRAMURAL RESEARCH 



procedures have also provided experimental data 
which indicate that tissue or cellular sites of anti- 
body synthesis are not equally radiosensitive. 
This suggests the possibility of using radiation as 
agent for the selection of certain sites of antibody 
production. It is also possible that the manifes- 
tation of immune reactions in vivo may depend 
on the participation of non-specific processes. 
The mast cell has been shown to play a role in 
the tissue reaction to the combination of antigen 
and antibody in tissue. Thus it becomes neces- 
sary to know the effects of radiation in this 
and other nonspecific events when measuring 
the effects of immune reactions in tissue. 

Dr. Maxwell continues his long-term studies on 
the effect of ionizing radiation on amino acids. 
His objective is to determine the mechanism of the 
chemical reactions induced by ionizing radiation 
in aqueous solutions of amino acids. These simple 
systems of biological interest result in information 
which may be applied to more complex biological 
systems, which is not easily available by direct 
investigation. From the simple system of aqueous 
amino acid, ionizing radiation has produced 13 
products which have been isolated and identified. 
Effects of temperature, pH and concentration of 
glycine are being studied. Only about one-half 
of the glycine is accounted for by these products. 
C"-glycine will be used to search for additional 
products. 

Dr. Riesz is also engaged in studying the effect 
of irradiation of in vitro systems. He is bombard- 
ing aqueuos solutions of acetone with cobalt 
gamma rays and studying the reaction products. 
He has isolated hydrogen gas, hydrogen peroxide, 
hydroxyacetone and 2,5-hexanedione. In dilute 
solutions the yields can be quantitatively explained 
in terms of the reactions of hydrogen atoms and 
hydroxyl radicals. In concentrated solution, the 
concept of hydrogen atom precursors is required 
to explain the observed results. 



RADIATION BRANCH 

The year 1961 has seen the separation from the 
Radiation Branch of the experimental radiobio- 
logical research components. 

In respect to service functions, the Radiation 
Branch is responsible for meeting the medical 



radiation therapy needs of the Clinical Center. 
As a measure of the size of tliis operation some 
434 courses of patient irradiation comprising 1,798 
irradiations were administei-ed and 202 niedical 
consultation requests were processed in 1961. 

Non-clinical irradiation services are also per- 
formed as a continuing responsibility of the 
Radiation Branch to meet the requirements of the 
NIH and, on request, to other agencies of the Fed- 
eral Government. The scope of this operation is 
very broad; 2,651 high and mediimi energ;^^ ex- 
posures being given to a variety of biological ma- 
terials, each exposure generally involving the 
simultaneous irradiation of multiple units, and 
2,100 animals have been irradiated for variously 
prolonged periods of time in a "low-level" irradia- 
tion facility. Thirty-six percent of these irradia- 
tions were performed for the operations of tlie 
Radiation Branch and 16% of the high-energy 
irradiations were performed for other government 
agencies. The remaining 40% were performed 
for other NIH laboratories. These irradiations 
are performed, largely, by the physics staff. 

The clinical research activities of the Radiation 
Branch can be grouped conveniently in two cate- 
gories, the one having to do with the definitive 
relief of human cancer and the other with the 
human patho-physiology of ionizing radiations. 
In the former belong the studies of the significance 
of the relationship of dose and time and related 
considerations in the cancerocidal effect of irradia- 
tion and the chondrosarcoma and the childhood 
cancer studies. 

The dose time studies have been extended to in- 
clude studies of tumor and patient i-esponses at 
extreme ends of the dose and time scales. The 
cases studied have been those of carcinoma of the 
upper food and air passages. Single irradiations 
with tiunor doses of the order of 2500 roentgens 
are being applied in order to have a basic experi- 
ence and to define some responses as a background 
for other clinical studies where, as in high- 
pressure oxygen radiotherapy, prolonged or multi- 
ple treatments may not be possible. These 
responses are being compared with those at the 
other extreme of the dose time scale, that is, those 
cases receiving doses of the order of 9000 r over 
100 days. Some of these cases are still being fol- 
lowed and many interesting observations have re- 
sulted. Among the more interesting conclusions 



NATIONAL CANCER INSTITUTB 



55 



that can be dra-wn from the observations is that 
dose-^e?' se, is critical at either extremity. Tliis 
material will be worked up in detail at an appro- 
priate time as several years are required to ac- 
cmnulate data of these sorts. In respect to 
chondrosarcoma studies, a method has been elabo- 
rated for the determiaation of the "volume of 
distribution" of sulfur and this can now be studied 
before and after; for example, amputation, resec- 
tion, or external beam or radioisotope radiation 
therapy. Extensive experience has now been 
gaiaed with the clinical, radiotherapeutic, and 
other aspects of the so-called undifferentiated solid 
cancers of childliood, especially those of the head. 
A very substantial experience has also been ac- 
cumulated in respect to the indications for and 
the responses to local irradiation of the iofiltrative 
local lesions of acute childhood leukemia. 

The detailed hematological responses and the 
recovery of radiation-injured hemopoietic ftuic- 
tions are being studies in patients receiving thera- 
peutic total body irradiation. This total body ir- 
radiation is being delivered at a faster rate than 
has been reported and the responses which have 
been observed suggest that there are real rate dif- 
ferences in the degrees of response to a given dose. 
A detailed study of the quantitative relationsliip 
of the prolongation of the time in which radio iron 
is removed from the plasma ( a measure of erythro- 
poiesis) to the dose of total body irradiation and 
the quantitative relationship of changes in the 
white blood cell mobilization response to bacterial 
toxin to the dose of total body irradiation is being 
undertaken. Eenal function studies are showing 
for the first time, by the use of certain refined tests 
of renal fimction, the changes which may occur as 
a result of therapeutic irradiation of the abdomen 
and renal regions for metastatic abdominal cancer 
and wliich changes are not ordinarily reflected in 
common tests of renal function. An observation 
of great interest is that changes are demonstrable 
after what would ordinarily be considered rela- 
tively small doses of radiation. Calcium metabolic 
radiation effect studies have so far largely been 
limited to calcium balance studies and some tracer 
studies with radioactive Calcium ". To a limited 
degree, we have been able to demonstrate local 
changes in calcium deposition in destructive neo- 
plastic bone lesions following radiation therapy. 
(J. E. Andrews) 



A quantitative mammalian cell radiobiology 
system has been devised. It is miique in that it 
iiomially is an anoxic one in respect to the oxygen 
effect. The full exploitation of the system has been 
handicapped by lack of adequate numbers of ani- 
mals, but, nevertheless, a substantial amount of 
quantitative information on the influence of cell 
population number, ionization density (LET), 
oxygenation, dose-fractionation, and chemical 
modification has been obtained. Most of this in- 
formation has a direct applicability to problems in 
clinical radiotherapy research and it points the 
way to directions along wliich such research should 
move. 



SURGERY BRANCH 

Local Wound Chemotherapy 

The effectiveness of local wound chemotherapy 
was studied in a number of systems. Using urine 
5-hydroxyindoleacetic acid content and time of on- 
set of palpable tumor of the P-815-MI ascitic mast 
cell tmnor as measures of tumor growth, previous 
animal studies with formaldehyde suggested that 
when the drug was applied to the "wound" prior to 
iiioculation with a tumor cell suspension, an in- 
creased growth of tumor was observed. Further 
studies tliis past year have shown that a true "pre- 
treatment" effect was not seen when an open womid 
was used, i.e., when an incision was made simulat- 
ing clinical surgical procedures. However, when 
an air bubble "womid" was created the tumor ap- 
peared, earlier, and an increase growth rate was 
observed. 

In other studies of local chemotherapy a 90- 
95% effectiveness of proflavine hemisulfate in pre- 
venting the "take" of S-91 melanoma or mast cell 
tiunor in open axillaiy womids was shown. This 
effectiveness was diminished to 48% if the wounds 
were made hemorrhagic previous to instillation of 
the drug. This would seem to be important in any 
clinical application of local chemotherapy. The 
bloody and serous drainage could "neutralize" any 
local di'ug. 

Clinical application of local chemotherapy has 
been under study for some time. At first, the op- 
erative wounds were sprayed with salme for 
cytologic studies. Later, a group of patients had 



56 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



their wounds treated with 0.5% formaldehyde. 
Followup studies of a group of head and neck and 
cervix cancers showed very little effect of the form- 
aldehyde in changing the incidence of local re- 
currences. However, as followup information 
becomes more complete, it would appear that local 
wound treatment may be of value. In a study of 
119 uterine cervix cancers that received radical 
surgical excision, there were 54 that received no 
wound treatment, 27 that had 0.5% formaldehyde, 
and 38 that had formaldehyde plus the use of 
picris acid-treated sutures. The numbers of ad- 
vanced cases were the same in. each of the 3 groups, 
and the proportion with positive wound washings 
was the same. The gross local recurrence experi- 
ence for the 3 groups was 21. of 54 (39%) for those 
with no treatment, 4 in 27 (15%) for the formal- 
dehyde group, and 10 of 38 (27%) for the com- 
bined treatment. At 30 months after surgery, 
when almost all the local recurrences had mani- 
fested themeselves, the cumulative percent with 
local recurrence, calculated by life table methods, 
was 49% for the no treatment gx'oup, 28% for 
picric acid group, and 17% for the formaldehyde 
treated patients. Since the numbers involved in 
each group are small, the significance is questioned. 
However, when the date is examined from another 
point of view, it takes on added significance. If 
the wound treatment results are correlated with 
wound washings, the cumulative percent with local 
recurrence at 30 months in 11 patients with posi- 
tive wound washings was 56% for those with no 
wound treatment, and 57% for those positive cases 
treated. The 23 patients with negative woimd 
washings and no treatment had 64% incidence of 
local recurrence, while the 40 treated negative 
cases had only a 15% incidence of local recurrence. 
This could be interpreted as a quantitative factor 
in wound contamination, that is, if the wound has 
a heavy contamination with tumor cells, enough 
to give positive wound washings, the locally ap- 
plied drug is not effective. The 58% cumulative 
local recurrence experience is the same for the en- 
tire group of positive washing cases. However, if 
the operative wound has only minimal contamina- 
tion so as to make it difficult to wash and identify 
cancer cells from a wound, the local drug treat- 
ment is able to kill these individual cells. 

It has been difficult to understand why patients 
with positive wound washings did not develop a 



significantly greater proportion of local recurrent 
cancer than those with negative washings. In the 
119 cervix cancers under study, wound washing 
studies were performed in 96. Twenty-three of 
these were positive for cancer cells. The local re- 
currence experience for this gi'oup was 9/23 ( 39 % ) 
for the positive washings, and 17/63 (27%) for 
negative washings. Tliis difference is not what one 
would expect, if the true picture of wound con- 
tamination were seen in the studj^ of wound wash- 
ings. (Smith) 

In addition to the re-evaluation of wound wash- 
ing results, wound drainage has been under study 
for the past 1% years. A review of 40 cases in 
whom both wound washing and womid drainage 
fluid was examined, revealed additional cases that 
had cancer cell contamination of their wounds. 
There were 8 with positive wound washings and an 
additional 4 in whom woimd washings were nega- 
tive, but wound drainage collected as late as 68 
hours after surgery contained cancer cells. This 
now means that calculation of local I'ecurrence 
rates in relation to wound contamination must be 
based upon 12 of the 40, and not 8 as in previous 
studies. This may accomit for some of the "false" 
negatives. 

In addition to evaluation of local recurrence 
rates based upon the above data, studies of circu- 
lating cancer cells shows a high incidence of cells 
in local blood draining a tumor. The role of cir- 
culating cancer cells in relation to the true degi-ee 
of wound contamination remains to be worked out. 

A satisfactory method has been developed for 
sampling blood from patients at regular intervals. 
Tumor cell embolization is greater between 6 a.m. 
and 4 p.m. Increased embolization during cer- 
tain phases of operating room eveiits was demon- 
strated. However, increased tumor cell "showers" 
during surgery is not uniform. It was noted that 
in 20 uterine cancer patients, a circulating tumor 
cell was found in 1 or moi'e speciments from 5 pa- 
tieiits. Life table method analysis suggests that 
the true prevalence of circulating tumor cells in 
peripheral blood in these patients was about 30%. 
The yield of "positives" for cancer of head and 
neck seems to be lower than uterine cancer. 

The circulatuig tumor cell studies have also 
made possible observations on the occurrence of 
megakaryocytes in blood. The range is variable, 
but in general is greater in blood from the inferior 



NATIONAL CANCER INSTITUTE 



57 



vena cava than in antecubital or jugular vein 
blood. As with tumor cells, there are greater 
numbers of megakaryoc3d;es found during the 
6 a.m. to 4 p.m. interval. The individual peak 
occurrence of circulating tumor cells and mega- 
karyocytes do not coincide. The numbers of these 
cells do not correspond to the extent of tumor 
present in the patient. Preliminary observations 
suggest that circulating megakaryocytes are ex- 
tremely sparse in normal individuals. 

Tumor Growth and Metastases 

A biologic test system to measure the rate of 
mitosis in the rapidly regenerating liver follow- 
ing partial hepatectomy in the rat has been de- 
veloped. The rate of cell growth has been meas- 
ured by means of dry weight, mitotic counts, and 
measurements of DNA synthesis by tritiated 
thymidine. Data accumulated to date indicates 
that serum from normal individuals contains two 
fractions; one capable of inhibiting cell growth, 
and the other capable of stimulating growth of 
the regenerating liver. Serum from patients with 
cancer contains a stimulating fraction, but does 
not contain an inhibiting fraction in the same 
concentration as does normal serum. The addi- 
tion of the hihibiting fraction of normal sermn 
produces inhibition of liver regeneration, indi- 
cating that the deficit in cancer serum can be re- 
placed. The periodic injection of inliibitmg sub- 
stance into animals with a transplanted tumor 
delays the time of appearance of the tumor, and 
slows its growth rate. 

It has been consistently found with five animal 
tumor systems that removal of a primary tumor 
decreased the number of lung metastases. The 
metastases which did develop in lungs after pri- 
mary tumor removal grew to a larger size than 
those which were found in comparable primary 
tumor-bearing animals. Complete amputation 
of the primary tumor sharply reduced the number 
of metastatic foci, while incomplete removal of 
the primary tumor did not significantly alter the 
number of foci. Wlien only part of the cancer 
was removed, results were similar to the unam- 
putated group. This suggested that surgical 
trauma fer se does not result in enhanced forma- 
tion of metastases. 



The effect of pregnancy on spread of tumor has 
been studied. Mated mice, whether pregnant or 
not, have larger primary tumors when sacrificed 
39 days after tumor inoculation. No clear effect of 
mating on the occurrence of lung metastases was 
noted, but growth of such metastases was found to 
be inhibited in pregnant mice. 

Blood of newborn mice, from mothers with 
S-91 melanoma, revealed cells which were char- 
acteristic of malignancy. Control blood from nor- 
mal fetuses did not contain such cells. It was 
noted that maternal pelvic organ metastases were 
not present in 2 of the 8 positive groups. Metas- 
tases were not evident in any of the offspring. 
Thirty-one of the placentas were recovered. Se- 
rial sections of placentas, fetuses, and newborn 
lungs failed to disclose any metastatic foci. 

Growth hormone increased primary tumor 
growth, carcass weight, and number of large 
pulmonai-y metastases in 2 animal tumor systems. 
Adrenocorticotropic hormone (ACTH) simul- 
taneously decreased tumor gi"owth, carcass weight, 
and number of large metastases. Wlien growth 
hormone and ACTH were administered together, 
only the effect of growth hormone was observed. 
The two drugs seemed to have an antagonistic 
action. Neither altered timior take, latent period, 
or growth rate of the primary or secondary tumor 
was not transferred when the primary tumor was 
transplanted to untreated animals. 

Virus and Surgical Treatment of Cervical Cancer 

During the past year the data obtained from 
the cervix virus study cases have been collected. 
Of special interest is the survival data in those 
patients in whom virus was injected into the cervix 
prior to excisional surgery. It is apparent that 
those patients who had virus placed in their cervix, 
and were then subjected to pelvic excisional 
surgery, the life survival curve was increased for 
the first 24^36 months after surgery. The cumula- 
tive percent surviving at 12 months of those who 
had Coxsackie virus plus definitive surgery was 
93%. Adenovirus and surgery gave an 88% sur- 
vival at 12 months, while for surgery alone there 
was 68% survival at 12 months. Statistical 
analysis shows that at 6 months the 35 patients 
with surgical excision and no virus had 74%, while 
22 virus-treated patients had 91% survival. 



58 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Head and Neck Cancer 

A study of 89 patients with invasive head and 
neck cancer revealed over 20% showing clinical 
evidence of distant metastases during the patient's 
life. At autopsy the incidence of distant 
metastases during the patient's life. At autopsy 
the incidence of distant metastases was 50% . This 
is far higher than generally recognized, and dem- 
onstrates the true "cancer" characteristics of these 
neoplasms. The overall degree of metastasizabil- 
ity of head and neck cancers is less than other 
cancers, i.e., in a cooperative study of 600 laryngeal 
cancers only 151 (25%) had lymph node metas- 
tases at the time of admission, none were below the 
clavicle. The overall five year survival rate was 
65%. Therapy for early stages gave good results, 
whether by radiation or surgery (91% for Stage 
1, 75%) for Stage 2, 45%o for Stage 3, and 22% for 
Stage 4) . Eadiation therapy was not of value in 
advanced stages. There were no survivors in Stage 
4 cancers treated by radiation, while surgical 
therapy resulted in a 34% five year salvage. Even 
bulky tumors when treated by surgical excision, 
resulted in 60% salvage. The important prog- 
nostic factor was the development of a lymph node 
metastasis. "When a lymph node metastasis was 
observed on admission, sui-vival rate was less than 
half that seen for similar primary tumors without 
node metastases. Only 30% of the 151 with node 
metastases survived five years. 

Cancer of Uterine Cervix 

A true contrast of cancer behavior is seen in the 
cervix study. The overall salvage of these cases 
seen at the Clinical Center was 37%. This study 
consisted of 206 cervix cancers. Twenty-two of 
these had their cancer arise in a cervical stump. 
There were 46 that were inoperable on admission 
because of extent of disease, medical status, etc. 
An additional 41 patients were explored and found 
inoperable. There were 37 radical hysterectomies, 
19 anterior exenterations, 63 total pelvic exentera- 
tions. Eighty-seven percent of the operable 
group had advanced disease (Stage C or higher). 
There were 7 operative deaths in the 119 cases 
(5.6%) . Six of these occurred in the total pelvic 
exenteration group. The causes of these opera- 
tive deaths are interestuag. One died on the oper- 
ating table of cardiac arrest. There was one mas- 



sive j)ulmonary embolus on the seventh postoper- 
ative day. One patient died on the eightli day 
after re-exploration for intestinal obstruction. 
The remaining 4 deaths were associated with 
septicemia, occurring as late as one month after 
exenteration and resulting from multiple com- 
plications associated with pre-existing pelvic in- 
flammatory disease, fistula formation, etc. Five 
of the 6 deaths in the exenteration group occurred 
in post-radiation patients. 

The cumulative percent surviving 5 yeai-s (life 
table method) for the hysterectomy group was 
57%. The same survival was noted in the 
anterior exenteration group. Five year sui^vival 
following total exenteration dropped to 17%. 
Survival was related to extent of disease, and not 
on the operative procedure performed. The ac- 
ceptance of total pelvic exenteration has depended 
upon the development of a satisfactory means of 
urinary diversion. The first exenterations done 
at this hospital were given a "wet colostomy." 
One-third of these few cases died free of cancer 
in 24r-36 montlis from renal failure secondary to 
infection. Another % rapidly died of their cancer 
before their renal failure became severe enough to 
cause death. This remaining group was either 
converted to an ileal loop, or died of their cancer. 
The loop method of urinary diversion is now 
utilized. We have had no incidence of renal fail- 
ure, and no deaths attributed to this type of 
urinary diversion. 

LABORATORY OF VIRAL ONCOLOGY 

The work of two of the three sections of the Lab- 
oratory of Viral Oncology, namely the Virus On- 
cology and Cellular Biology Sections, has been in 
the area of viruses in relation to cancer, althougli 
the additional areas of (1) carcinogenesis asso- 
ciated with nonviral agents, (2) ultrastructure 
(electron microscopic) of various types of cancer 
and normal cells, (3) cancer immimology, and 
(4) radiation have also been embraced. The M'ork 
of the Tissue Culture Section includes studies 
with tissue cultures of the phenomenon of malig- 
nant transformation m vitro^ the malignant cell, 
and the normal cell from wliich it arose. j\Iuch 
work has been done also on the development of 
tissue culture methodology and on the applications 
of newer methods to the studies mentioned. 



NATIONAL CANCER INSTITUTE 



59 



I. Viruses in Relation to Cancer 

a. Laboratory Animal Systems 
1. MuEiNE Leukemia Viruses 

a. Moloney virus: Dr. Moloney lias contmued 
his studies -with tlie virus he discovered in 195S, 
and first reported upon in 1959. His studies on 
animal specificity have been extended to include 
(1) a total of 8 strains and 5 hybrid gi'oups of 
inbred laboratory mice, (2) wild mice (in collabo- 
ration with Drs. Andervont and Duiui), (3) two 
strains of rats, (4) hamsters, (5) guinea pigs, (6) 
rabbits, and (7) sub-human primates (6 rhesus 
and 1 cynomologus monkeys). All types of mice 
and rats tested, including wild mice, are suscepti- 
ble to the Moloney virus. Hamsters also devel- 
oped neoplasms of the reticular system, diagnosed 
tentatively by Dr. Thehna Dunn, as reticulum cell 
sarcoma. The first hamster tmnor appeared 12 
montlis after inoculation of the virus neonatally, 
and at 14 months the incidence among different 
groups of hamsters varies from 12.5 to 33%. 
These virus-induced neoplasms are readily trans- 
plantable in hamsters and studies are now under 
way to determine whether the virus is recoverable 
from the original and transplanted neoplasms. 
No lesions have as yet developed in other species. 
The studies in monkeys were started only recently 
and have been in progress for only a few months. 

Further additional information of importance 
has been obtained by Dr. Moloney on the stability 
of Ms virus and on its chemical, physical and 
biological properties. Of special interest is the 
fact that it is inactivated by heating at 56° C for 
30 minutes, that is, at a temperature slightly lower 
than that used for pasteurization. Significant 
contributions have been made by him also to a 
number of collaborative studies to be reported 
upon by others. One ]oint study, in collaboration 
with Dr. Dalton, has led to some of the most im- 
portant developments of the year. Here, the con- 
tributions of each of these investigators were 
interdependent and of equal import in the devel- 
opments to be described. 

Pursumg the finding reported last year of an 
abimdance of virus particles in megakaryocytes of 
the bone marrow of animals infected with the 
Moloney vii-us, electron micrographic studies were 
made of the blood platelets, which derive from 

643351—62 6 



megakar3-ocyi;es. Platelets were found to contain 
characteristic virus particles, but what was more 
significant was the presence of free virus particles 
in spaces between the platelets in the specimens 
examined mider the electron microscope. This led 
to fractionation of larger volumes of blood and 
separation by ultracentrifugation of the "micro- 
some" fraction, which would be expected to in- 
clude any virus. Duplicate pellets of sedimented 
material were used for electron-microscopic 
studies by Dr. Dalton and for biological tests in 
mice and rats by Dr. Maloney. Both procedures 
confirmed the presence of large amounts of leu- 
kemia virus in the fractions derived from blood. 
The biological studies indicated a yield of virus 
about 10 times higher than previously had been 
obtained from leukemic-tissue sources (on a gram 
equivalent basis), and the electron micrographs 
indicated that the virus preparation derived from 
blood was in a fairly high state of purity with 
respect to formed elements, there being little 
else but virus particles and some debris, probably 
of platelets. 

These results led to the following further im- 
portant developments: (1) Dr. Moloney was able 
to modify and refine his differential centrifugation 
procedures so that apparently very high purifica- 
tion of the virus was achieved, as judged by elec- 
tron microscopy. TMs acliievement is comparable 
to that reported earlier by Beard and his associates 
for the virus of fowl myeloblastosis, wliich also 
is present in high concentration in the blood of 
infected fowls, and wliich can be separated from 
the blood in practically pure form by the simple 
procedure of differential centrifugation. Tliis 
development makes it possible, and work has 
already been initiated to produce relatively large 
quantities of higlily purified virus for chemical, 
physical and biological studies, as well as for 
large scale use in producing leukemic animals for 
chemotherapy research and screening, and for 
studies on vaccine production. 

(2) The consistency of the association between 
biological activity and the presence of virus parti- 
cles in electron micrograj^hs has added a valualile 
tool not only for studying the pathogenesis of the 
disease in animals (e.g., prior to the appearance of 
lesions) , but also has opened a door to the human 
problem by providing a fairly simple and practical 
procedure for the potential detection and isolation 



60 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



of human leukemia viruses, if such exist, compa- 
rable to this murine virus. 

Using improved lead staining procedures in his 
electron micrographic studies. Dr. Dalton has 
found the mature Moloney virus particle to meas- 
ure 97.5 millimicrons in diameter. The particle 
is bound by a double limiting membrane, the inner 
measurement of which is 8 millimicrons in diame- 
ter. A central, more electron dense nucleoid 
measures 65 millimicrons in diameter. Similar 
studies and determinations of particle size were 
made imder identical conditions on four additional 
murine leukemia agents. Thin section studies of 
two separate radiation induced leukemias of mice 
showed virus particles similar to those of viral 
leukemia in one but not in the other. Characteris- 
tic particles were also found in the megakaryocytes 
of bone marrow of leukemic mice of two different 
strains which develop the disease spontaneously. 
In all cases in which the particles were observed, 
they were found budding from the plasma mem- 
brane of malignant lymphoblasts or from intra- 
cellular membranes of megakaryocytes. 

Dr. Merwin has used the transplantable S 37 
mouse tumor known to carry the Moloney virus 
(originally isolated from S 37) to test her hy- 
pothesis that viruses in tumor tissue placed inside 
Algire diffusion chambers (which retain colls but 
allow diffusion of virus), and then placed intra- 
peritoneally in test mice, might be protected from 
host resistance and therefore have a better chance 
to adapt to a new host. Since virus would con- 
tinue to be produced by the living tumor cells 
within the chamber it might be possible to detect 
virus in tumors which, at a given time, is too low 
in concentration to be detected by other methods. 
Dr. Merwin's experiments are still in progress but 
a fairly high incidence (about 20%) of leukemia 
and Hodgkins-like lesions have already developed 
in the BALB/c hosts. These tumors come vip 
much earlier, and are to be distinguished from the 
sarcomas and plasma cell tumors which sometimes 
result from the implantation of the blank chambers 
alone. Similar studies with other virus-associated 
tumors of mice are under way. Dr. Merwin now 
plans to use this technique to test human tumor 
for their ability to induce characteristic lesions in 
mice. 

Dr. Manaker has previously reported the suc- 
cessful propagation of the Moloney virus in pri- 



mary tissue cultures of cells derived from mouse 
spleen. The cells which grew out under the condi- 
tions employed, and which supported growth of 
the virus, were amoeboid cells, probably of the 
reticular or macrophage type. Dr. Manaker has 
now succeeded in establishing a continuous Ime of 
these cells in tissue culture and has f omid them to 
be suitable for propagating the Moloney virus. 
This makes possible the large scale in vitro produc- 
tion of the Moloney virus m certified cell lines, 
without danger of infection with extraneous vi- 
ruses as might occur when primary cultures pre- 
pared from many mouse spleens are used. 

&. Manaker (O-60) virus: Dr. Manaker has 
isolated a new strain of mouse leukemia virus 
which differs in certain electron micrographic and 
biological properties from other murine leukemia 
viruses previously described. The isolate was 
picked up in tissue culture studies in which at- 
tempts were being made to propagate the elusive 
Schoolman-Schwartz agent isolated from a Swiss 
mouse leukemia. Whereas the latter agent iiiduces 
a solitary mesenteric tumor nodule as the charac- 
teristic lesion, the new strain isolated by Dr. Man- 
aker produces a generalized leukemia. Another 
difference is that active virus could not be extracted 
from the mesenteric tumor nodule by Schoolman 
and Schwartz and their associates, but only from 
the brains of mice bearing the induced tumor. Dr. 
Manaker's virus on the other hand, can be readily 
extracted from all involved tissues of the general- 
ized leukemia. Since the Schoolman-Schwartz 
agent is in the background of tlie picture, Dr. 
Manaker concludes that his virus is a derivative, 
or strain of that agent. The possibility remains, 
however, that the two are not related and that Dr. 
Manaker has discovered a new virus in the growing 
family group of murine leukemia vinises. Ex- 
tensive serological and immunological studies will 
be required to answer this question. 

The Manaker vii'us reaches a high concentration 
in leukemic tissues, comparable to the Moloney 
virus. Dr. Manaker has made extensive studies 
on the character of his virus and has now estab- 
lished it as a new laboratory model available for 
the study of neoplasia. 

c. Rausclmr virus: Dr. Rauscher has also dis- 
covered a new murine leukemia virus (designated 
as E-NCI-2) which differs dramatically from any 
hitherto described. Again, the Schoolman- 



NATIONAL CANCER INSTITUTB 



61 



Schwartz agent was in the background. Experi- 
ence with that agent had shown that most experi- 
mental attempts to pass it by means of cell-free 
filtrates fail, and only occasionally was success 
achieved. Wlien successful, the tumors appeared 
within 3 or 4 weeks, or not at all. 

In a large scale attempt to get at the factors 
associated with the elusiveness of the Schoolman- 
Schwartz agent, Dr. Eauscher inoculated a total 
of 508 test mice. None of them developed lesions 
within the 3 to 4 week period. However, after 11 
weeks one of this number developed a solid sub- 
cutaneous tumor at the site of needle penetration 
of the skin. This has not previously occurred 
with the Schoolman-Schwartz agent. The solid 
tumor was carried by transplantation through 9 
passages in BALB/c mice and attempts were then 
made to isolate a virus. A virus was isolated 
which, when inoculated into suckling mice, causes 
a tremendous enlargement of the spleen, up to 
60 times the size of normal spleens, within a period 
of about 15 days. Death results within about 35 
days. Sections of such spleens show large num- 
bers of nucleated red cells as well as undifferen- 
tiated cells of the white cell series. Peripheral 
blood counts show 28,000 to 250,000 nucleated cells 
per cc. Anemia is not present. Depending on the 
conditions of the experiment (dose, age at inocula- 
tion, etc.), some mice do not develop the initial 
spleen enlargement, and others which do develop 
large spleens survive beyond the 35-day period. 
In such animals a second syndrome appears which 
includes all of the manifestations of generalized 
leukemia, including thymic and peripheral lymph 
node enlargement. This second syndrome is fol- 
lowed by a second mortality peak. Virus isolated 
from animals which develop the early syndrome 
only and die early, or virus from animals which 
develop only the later syndrome (typical leu- 
kemia) and die after several months, is in both 
instances still capable of inducing both types of 
response. It appears therefore that both diseases 
are produced by one virus. 

Dr. Thelma Dunn is studying the pathogenesis 
of this agent and has determined the second syn- 
drome as a true leukemia, probably originating in 
the thymus. It has been tentatively classified by 
Dr. Dunn as a stem-cell leukemia, and is distinct 
from any other murine viral leukemia heretofore 
reported. 



The early enlargement of the spleen grossly 
resembles the Friend virus disease. However, the 
early syndrome produced by the Eauscher virus 
differs from the Friend disease in the following 
important respects: (1) the high incidence of 
true leukemia (80-100%) in mice which survive 
early death due to erythrolastosis ; (2) the early 
and continuing presence of greatly elevated levels 
of nucleated cells in the peripheral blood; (3) 
ED50 end titers up to 10"^ and 10"' within 70 to 
90 days; (4) the relative ease with which frag- 
ments of spleen, thymus and lymph nodes give 
rise to solid tumors at localized sites on trans- 
plantation, (5) the high susceptibility of mice of 
many different strains (the Friend virus grows 
in only 2 strains) ; (6) the early severe viremia 
of infected mice; and (7) clear differences in 
histopathology of the two diseases. 

The virus also differs antigenically from both 
the Friend and the Moloney viruses, since anti- 
sera against neither will neutralize it. 

The Eauscher virus will infect adult, as well 
as suckling mice and thus far, 9 different strains or 
crosses of inbred mice have been found to be sus- 
ceptible to it. 

The rapidity of appearance of palpable lesions 
(enlarged spleens), that is, within 5 to 15 days, 
makes bioassay of tliis virus much more practical, 
and less costly than for any other murine leukemia 
virus (which require several to many months). 
Chemotherapeutic studies on tumor viruses are 
also more practical with this agent, and collabora- 
tive studies with Drs. Chirigos and Boldin have 
already been initiated. 

Another new and somewhat strange member has 
been added to the family of murine leukemia 
viruses. 

d. Breyere-Moloney virus: The isolation of a 
new virus which produced a generalized leukemia 
in BALB/c mice was reported last year by Doc- 
tors Breyere and Moloney. The biological ac- 
tivity of this virus has been enhanced during the 
past year by serial selective passages and further 
studies have been carried out on animal suscepti- 
bility. Both C3H mice and Osbome-Mendel rats 
have been shown to be susceptible to this agent. 
2. Polyoma Viktjs 

Dr. Sarah Stewart is continuing her studies on 
the polyoma virus which she isolated in collabora- 
tion with Dr. Bemice Eddy several years asro. 



62 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Since this virus is the only known agent which 
has the combined properties of proclucmg tumors, 
causing cell lysis, and producing hemagglutma- 
tion of red blood cells, Dr. Stewart has been at- 
tempting to determine whether all of the proper- 
ties are possessed by a single virus particle, or 
whether more than one type of particle may be 
involved. 

In collaborative studies, Dr. Robert Cramer of 
the Pasteur Institute, Paris, has separated Dr. 
Stewart's virus preparations mto various electro- 
phoretic fractions and return them to Dr. Stewart 
for further study. Although earlier work on this 
project previously reported showed that the virus 
could be separated from inhibitors of the hemag- 
glutinating property by zone-electrophoresis, the 
continued studies have produced no evidence to 
date that more than one type of viral particle is 
involved in the three reactions, but the data are 
not yet conclusive. 

In other collaborative studies with Dr. M. Ida 
of the M. D. Anderson Hospital, Houston, the 
ability of the polyoma virus to cross placental 
barriers in mice and hamsters is being investi- 
gated. Offspring of mothers which had been 
inoculated at various stages of gestation were 
taken by caesarian section and raised by foster 
nursing on non-infected mothers. At the end of 
12 months, surviving test mice were divided into 
2 equal lots and one lot was treated with 150 r of 
total body radiation for 4 successive times at 3-day 
intervals. The other lot was not given any addi- 
tional treatment. None of the offspring of in- 
fected mothers, neither mice nor hamsters, 
developed tumors of the polyoma virus spectrum, 
indicating that the polyoma virus does not pass 
the placental barrier, at least in sufficient amounts 
to induce neoplasia. Antibody titers for the de- 
tection of subclinical infection with the polyoma 
virus could not be studied simultaneously in this 
experiment due to the lack of isolation facilities 
and the presence of other infected animals in the 
same room. 

Dr. Stewart's assistant, Mr. Jolm Landon, is 
studying the influence of fluorocarbon on lung 
tumor induction in hamsters by polyoma virus. 
Fluorocarbon was introduced intravenously into 
18-day-old hamsters and two days later they were 
inoculated intratracheally with polyoma virus. 
Control animals received only fluorocarbon intra- 



venously or only polyoma virus intratracheally. 
Several of the animals which received polyoma 
virus only developed Imig tumors, but many died 
from liver hemangiomas. The animals which 
were inoculated with both fluorocarbon and poly- 
oma virus had many more lung tumors which at- 
tained a larger size. Many of them were squa- 
mous cell carcinomas. No lesions were produced 
by fluorocarbon alone. 

Mr. M. B. Melroy continues to perform the valu- 
able service of running an extensive program for 
testing for the presence of polyoma vims antibody 
in mice used for experimentation in tumor-Aarus 
I'esearch. This service is under the general super- 
vision of Dr. Fink. 

3. Eotjs Sarcoma Vibtjs 

Doctors Eauscher and Fink are continuing their 
studies using the Rous sarcoma virus model in at- 
tempts to develop more sensitive methods for the 
detection of tumor virus and, perhaps, making 
possible the detection of virus in tmnors not now 
known to be virus induced, including human 
tumors. That the use of Freund's adjuvant in 
association with virus inoculation enhances the 
response to Rous sarcoma virus was reported last 
year, and this technique was introduced into the 
screening program for the detection of human 
viruses by the use of newborn mice (see section on 
human studies (B) ). The studies with Rous sar- 
coma virus are now nearing completion and it has 
been established that: (1) with test chickens pre- 
treated with Freund's adjuvant before inocula- 
tion of the virus, large granulomas appear within 
4 to 5 days. Inoculation into the granulomas of 
veiy small doses of Rous sarcoma virus (e.g. 0.01 
ED50) which ordinarily do not produce tumors or 
result in recoverable viras from inoculated hosts, 
caused the granulomatous lesions to grow rapidly, 
become tmnorous, and invade surrounding tissues. 
Virus was recoverable from such lesions in rela- 
tively high concentration (10- to lO* ED50 per 
gram). The technique therefore permitted 
propagation and recovery of virus in fairly high 
yields from subliminal doses, which luider ordi- 
nary conditions would not have led to tumor for- 
mation or recoverable virus. 

Wlien the adjuvant was injected at a later date 
into chickens which had previously received, but 
failed to respond to subliminal doses of the viinis 



NATIONAL CANCER INSTITUTE 



63 



by developing tumors, relatively high titers of 
antibody against the virus appeared. Thus, it 
was possible with tliis teclmique to establish that 
chickens were infected with subclinical doses of 
Eous sarcoma virus, although tmnors did not ap- 
pear within the usual latent period (35 days). 
Wliether tumors would appear after prolonged 
periods (e.g. many months) remains to be deter- 
mined. This is an important question since it in- 
volves the possibility that Freund's adjuvant may, 
in time, activate latent infections with subclinical 
doses of virus. 

As reported last year, the Eous sarcoma virus 
could be propagated serially in post-hatched Jap- 
anese quail, but not on the chorioallantoic 
membrane of the quail eggs. These studies, in 
collaboration with Dr. James A. Keyniers, have 
been continued and it has now been found that 
after ten passages in post-hatched quail, a "quail 
adapted" strain of Rous sarcoma virus has been 
established wliich can be propagated on the chorio- 
allantoic membrance of embryonated quail eggs. 
During twelve passages in quail eggs there was 
a gradual increase in the ability to grow in quail 
eggs, with a concomitant decrease, and finally 
complete loss, of the ability to grow also on the 
chorioallantoic membrane of embryonated chicken 
eggs. Thus a new, quail-egg adapted, strain of 
the Eous sarcoma virus has been established which 
will permit more extensive and more economical 
studies on host-virus interaction in miniature 
fowl hosts. 

4. OTHER VIRUSES IMPORTANT TO TUMOR ^TRUS 
RESEARCH 

a. Indigenous viruses 

(1) ZiEGEL Agents: Dr. Ziegel reported last 
year the discovery of a virus-like agent which he 
found budding from acinar cells of the pancreas 
in apparently normal chickens. The particles had 
all of the characteristics, including their forma- 
tion by budding from plasma membranes, of the 
known tumor viruses in both fowls and mice. It 
appeared therefore that Dr. Zeigel's agent might 
be a latent tumor virus, possibly the lymphoma- 
tosis virus which is known to be present, at least 
as a subclinical infection, in most strains of chick- 
ens. Dr. Ziegel has carried his electromicro- 
scopic studies further and, in collaboration with 
Dr. Rauscher who is making parallel biological 
studies, has found his agent budding from pancre- 



atic cell in chickens from flocks known- to be in- 
fected with lymphomatosis virus (from antibody 
studies), whereas he has yet to observe this phe- 
nomenon in cliickens of a strain obtained from Dr. 
B. R. Burmester (U.S.D.A., East Lansing, Mich- 
igan) which has a very low incidence of the dis- 
ease. By studying the offspring of hens showing 
serological evidence of infection with lymphoma- 
tosis virus. Dr. Zeigel has obtained additional 
evidence that his agent may be identical with the 
lymphomatosis virus. If tMs is the case, then Dr. 
Zeigel has discovered a very important, probably 
the most essential, site of replication of this virus 
in host tissues and has opened the door to early 
pathogenesis studies on this important disease of 
fowls. In addition to the possibility of control- 
ling this very important economical problem 
through greater knowledge of pathogenesis of the 
etiological agent, the understanding of this disease 
in chickens will contribute greatly also to the va\- 
derstanding of viral neoplasia in general. With 
the recent introduction of in vitro methods for 
quantitative assay of the lymphomatosis virus by 
Rubin (based upon interference with the Rous 
sarcoma virus) this fowl tumor virus model has 
become one of the most important laboratory 
models for studying the natural history and path- 
ogenesis of tumor viruses. 

(2) EJLHAM ("K") Virus: Dr. Ziegel is also 
carrying out electronmicrogi'aphic studies in col- 
laboration with Dr. Dalton on the "K" virus of 
Kilham, which is indigenous in certain strains of 
mice. He has found that the ultrastructure of this 
agent is similar to and suggests at least a morpho- 
logical relationship to the polyoma virus. 

(3) SIanakek Hepatitis Virus: Dr. Manaker 
has completed his studies on characterization of 
the indigenous hepatitis virus of BALB/c mice 
which he isolated in 1959 and reported upon last 
year. The general characteristics of this virus 
(designated ]\IHV-59) indicate membership in 
the "hepatoencephalitis group" of viruses. Sero- 
logical tests suggest a close relationship to one 
member of this group. The virus was found to 
produce widely disseminated inapparent infection 
in experimental mice. Precise quantitative meth- 
ods for in vitro assays of the virus, and serum 
neutralization methods for the detection of anti- 
body developed during the course of these studies 



64 



ANNUAL REVIEW OF rNTRAMXJRAL RESEARCH 



should, aid in the detection of tliis contaminating 
virus and lead to its elimination from defined 
colonies of experimental mice. 

B. Viruses in Human Neoplasias 

1. HUMAN CANCEROUS TISSUES 

a. Specimens from, surgical operations: Can- 
cerous tissues of various types removed at surgical 
operation are obtained through the cooperation 
of the U. S. P. H. S. Hospitals at Baltimore, Md., 
and Staten Island, N.Y. The specimens are 
frozen immediately upon removal and stored in 
low temperature chests ( — 50° C. or below) until 
used. For study, the specimens are homogenized 
and extracted in citrate buffer and the "micro- 
some" f I'action separated and concentrated by dif- 
ferential ultracentrifugation. Aliquots of this 
fraction, which would be expected to contain any 
viruses present, are used for a variety of proce- 
dures designed to demonstrate the presence of 
viruses and to propagate them in the laboratory. 
These procedures include : (1) Direct examination 
of the primary fraction imder an electron micro- 
scope (2) inoculations into various human and 
other cell lines in tissue culture; (3) inoculations 
into newborn mice; (4) inoculations onto chorio- 
allantoic membranes of embryonated chicken and 
Japanese quail eggs; (5) tests for ability to inter- 
fere with laiown viruses when inoculated into mice 
and embryonated eggs; (6) extraction of nucleic 
acid fractions and testing them in animal and tis- 
sue culture systems ; (7) similar secondary tests in- 
cluding all of the above (1-6) on fluids and cells 
from tissue cultures which received the primary 
inoculations. 

The following investigators are participating in 
these joint studies; Doctors Dal ton, Dunn, Fink, 
Manaker, Moloney, Rauscher, S. Stewart, and 
Zeigel, with the assistance of Miss Calnan, Mr. 
Kvedar, and Miss Valentine. 

Ten experiments initiated in calendar year 1960 
have reached the critical observation period during 
the current year. They involved the following 
human tumor specimens: 4 carcinomas of the 
stomach, 3 carcinomas of the breast, 1 carcinoma 
of the colon, 1 carcinoma of the lung, and 1 
sarcoma of the pelvis. 

In one of these experiments, involving a carci- 
noma of the stomach, 21.5 percent of the mice in- 



jected with the microsome fraction when newborn 
have developed neoplasms, whereas none of the 
control mice in the same experiment have devel- 
oped such lesions by the 13th month of the obser- 
vation period. Of 11 tumorous animals out of a 
total of 51 inoculated in the test groups, 9 have 
developed bilateral kidney carcinomas, a type of 
cancer which has never been observed to occur 
spontaneously in the strain of mouse employed, 
i.e., BAXiB/c. Significant also is the fact that 
such tumors have been observed to occur only very 
rarely among laboratory mice of all strains. In 
addition to the kidney timiors, several of the mice 
also had primary tumors of the pancreas and liver. 
The mice have been tested and shown to be free of 
polyoma virus infection. (Kidney carcinomas 
have not been observed with the polyoma virus.) 

The studies involving newborn mice are under 
the direction of Dr. Fink. In one of the two test 
groups which received the microsome fraction of 
the gastric carcinoma, Freund's adjuvant was 
added to the inoculums by Dr. Fink. The tumors 
appeared several weeks earlier in this group than 
in the group receiving straight microsome frac- 
tion (see also studies on animal systems, under 
Rous sarcoma). (A(3)). No tumors have oc- 
curred with adjuvant alone, with buffer alone, or 
among the iminoculated controls. 

These results confirm those of 5 other labora- 
tories which have reported the induction of tu- 
mors in mice injected neonatally with extracts or 
fractions of human tumors. They differ from 
the results of others, however, in that the pre- 
doninant type of tumor induced was not one which 
sometimes occurs spontaneously (with low fre- 
quency) in the strain of test mice employed. 

No significant results were obtained in the other 
9 experiments of this series. Although tumors of 
the types that BALB/c mice develop spontane- 
ously with low frequency as they grow old (e.g. 
lung tumors and leukemia) are now appearing 
among the older mice (14 to 16 months) in several 
of the experiments, such tumors occur with equal 
frequency among both test and control mice. 

The positive results reported from other lab- 
oratories have been interpreted either as: (1) the 
induction of tumors in mice by tlie replication and 
direct action of a human viral agent, or (2) the 
enhancement, or activation of a latent mouse tu- 
mor virus by some factor in human tumors, prob- 



NATIONAL CANCER INSTITUTE 



65 



ably viral in nature. In either event, the possi- 
bility exists that newborn mice may serve as a 
valuable biological iiadicator for the detection of 
human tumor viruses. The present series of ex- 
periments was for the purpose of further investi- 
gating this possibility. 

Even a single positive experiment among the 
10 which have been carried out justifies a more 
extensive investigation of tliis potential test sys- 
tem. Such studies have already been initiated 
under a contract with a private industry. 

No other evidence of a virus associated with 
the human specimens has been obtained with the 
other methods employed in this series of 
experiments. 

&. Specimens derived at autopsy: A single 
large experiment involving a total of 300 BALB/c 
mice (50 in each of 3 test and 3 control groups) 
was carried out in an attempt to repeat the 
studies of Schwartz et al. in which it was re- 
ported that microsome fractions of brain removed 
at autopsy from human leukemia subjects were 
capable of inducing leukemia in test mice. The 
strain of mouse used by Schwartz and associates 
was Swiss albino. 

The methods employed in the present study 
were the same as described by Schwartz (Blood 
15: 758-760, 1960), except that a different strain 
of mice (BALB/c) was used. 

No leukemias were induced in BALB/c mice 
within a period of a few weeks as reported by 
Schwartz et al. for Swiss mice, and no significant 
tumor induction has occurred among mice of this 
experiment within a 12 month observation period. 

2. Human Leukemic Blood and Bone JSLierow 

As a result of the significant findings with a 
murine leukemia virus model and the develop- 
ment of practical methods for the examination 
of blood and bone marrow described in another 
section of this report {Murine leukemia^, Moloney 
virus (11(A) ), Doctors Dalton and Moloney car- 
ried out exploratory studies on human leukemic 
specimens using the teclmiques worked out with 
the animal system. Briefly, the method consisted 
in the fractionation of citrated blood, or of bone 
marrow homogenates in citrate buffer, by differen- 
tial centrifugation and concentration of the 
"microsome" fraction by ultracentrifugation. 
Keplicate pellets of the concentrated materials 



were then fixed for thin-section electron micros- 
copy, used for inoculation into newborn mice, or 
stored at low temperature for future use. 

The experiments have been in progress for only 
5 months, and none of the test mice have as yet 
developed neoplasms. 

Out of a total of 14 human leukemia cases 
studied thus far, 11 specimens have revealed virus- 
like particles under the electron microscope. In 
some of these cases a careful search had to be 
made in order to find an occasional characteristic 
particle, but in several of the specimens many 
particles having a morphology similar to murine 
and fowl leukemia virus particles, could be seen 
within a single field under the electron micro- 
scope. Other replicate pellet suspensions from 
such cases were turned over to the other membei-s 
of the laboratory who are collaborating on tliis 
problem, for attempts to propagate the agent in 
the laboratory. In particular, efforts to propa- 
gate a virus in various types of human cell lines 
in tissue culture are being made by Doctors Man- 
aker and Stewart. 

It should be emphasized that the finding of viral 
particles does not necessarily indicate that they 
are etiologically related to the human neoplastic 
disease, since they could represent extraneous, 
or contaminating "passenger" viruses. For de- 
termining this possibility it will be necessary 
to propagate each such virus isolate m relatively 
large quantities in the laboratory so that exten- 
sive serological, immunological and biological 
studies can be carried out for identifying or char- 
acterizing it. Looking toward tliis end a viral 
diagnostic laboratory, as well as other "service" 
laboratories, have been set up on the Bethesda 
campus, and off-campus facilities for producing 
and testing large quantities of virus have been 
activated through contracts with private indus- 
try. In addition to virus production for charac- 
. terization studies, exploratoiy studies on vaccine 
production will also be undertaken in the off- 
campus facilities. 

The suggestive preliminary findings have also 
stimulated a closer collaboration between the lab- 
oratory and clinical branches of the N.C.I., and 
arrangements have been made through Dr. Emil 
Frei for the obtaining of an abimdance of clinical 
specimens. Dr. George Porter is acting as liaison 
between the laboratory and clinical groups, and is 



66 



A]SnsrUAL REVIEW OF INTRAMURAL RESEARCH 



participating in both the laboratory and clinical 
research. 

Specimens are also being received from field 
cases of leukemia, particularly from such "cluster" 
areas as Niles, 111., through the cooperation of the 
Epidemiology Branch of the Field Studies Divi- 
sion. Doctors L. Zelkowitz and Donald Arm- 
strong of this Branch have been collaborating 
with Dr. Moloney, Dr. Stewart and Mr. Culler in 
the procurement of field specimens. 

3. Othee Human- Lesions 

Another type of human disease being investi- 
gated by Dr. Sarah Stewart with respect to a 
possible viral etiology is the Histiocytosis X group 
of diseases. The gross pathological manifestations 
of the proliferative stages of these diseases are 
sometimes confused with early neoplastic lesions, 
particularly of the bone. Dr. Stewart received a 
biopsy specimen from a case of eosinophilic gran- 
uloma in a 20-month child, with the provisional 
diagnosis (before frozen section histological 
diagnosis) of osteosarcoma. Dr. Stewart inocu- 
lated tissue cultures with the biopsy material and 
was successful in propagating a filterable agent 
which appears to be a virus. Further eiforts to 
characterize it are in progress. The agent pro- 
duces cytopathogenic changes in cells in tissue 
culture, ajtid subcutaneous granulomatous lesions 
and hydrocephalus when innoculated into new- 
bom hamsters. There is no proof as yet that the 
agent which has been isolated is related etiolog- 
ically to the human disease, but through collabora- 
tion with Dr. Robert Andrews and other members 
of the staff of the Radiation Branch of NCI, other 
cases of the histiocytosis X group of diseases are 
being admitted to the Clinical Center for further 
attempts to isolate similar agents and determine 
their relationship to etiology. 

II. Tissue Culture 

A. General 

The outstanding contributions of members of 
the Tissue Culture Section to the field of tissue 
culture, both as a method of approach and as a 
new branch of cellular biology, are well known. 
The work of this section continues to be of the 
same high quality and step-wise progress continues 
to be made on several difficult problems of impor- 
tance to cancer research that have been embraced 
during recent years. 



The use of short-term tissue cultures in various 
branches of biomedical research and routine labo- 
ratory testing, particularh' in virologj', is now so 
extensive and so commonplace that, to many, the 
term "tissue culture"' has come to represent a 
method rather than a field of research. The ulti- 
mate goals of achieving truly long-term mainte- 
nance of manunalian cells in vitro, while preserv- 
ing their normal states and physiological proper- 
ties so that life processes may be investigated at 
the intracellular level, requires continuing basic 
research on cell biology and on factors associated 
with cell homeostasis. It is such studies, directed 
toward such goals, with which the research of tlie 
Tissue Culture Section is primarily concerned. 
Perhaps tlie time has come when tliis field of en- 
deavour should be given some other label, e.g., In 
Vitro Cell Biology. 

B. In vitro Cell Biology Studies 

1. NtJTEITIONAL AND MeTABOLIC. DoctOFS EvaUS, 

Sanford, Westfall, and Bryant and their asso- 
ciates are continuing their studies on the nutri- 
tional requirements of cells of various types and 
species of origin. A completely chemically de- 
fined, protein free, culture medium which 
sustains the growth of several lines of cells was 
reported last year. Several additional cell lines 
have been adapted to the completely defined me- 
diimi durmg the past year, of particular impor- 
tance being two sub-lines of Gey's HeLa strain of 
cervical adenocarcinoma which is so widely used 
in cancer and virus research. Further extensive 
studies have been made on tlie activity of the 
vitamins and reducing agents in the medium. 
These studies led to the first clear-cut demonstra- 
tion that biotin is required for survival of mam- 
malian tissue cells in vitro. Also, the addition of 
serum protein to an otherwise chemically defined, 
but controlled vitamin and amino acid deficient, 
medium was found to prevent development and 
manifestation of certain vitamin and amino acid 
deficiencies. It appears tlierefore that serum pro- 
tein contributes appreciable amounts of vitamins 
and amino acids to cells. By supplying appropri- 
ate quantities of such required components, a fur- 
ther improvement has been made in the com]-)letely 
chemically defined, protein free, medium but some 
lines of cells still cannot be adapted to it. Much 
work still needs to be done in the development of 
this important problem. Of particular impor- 



NATIONAL CANCER INSTITUTEi 



67 



tance, and now being emphasized, is the adaptation 
of stable lines of cells which support virus propa- 
gation, for eventual use in the mass production of 
vaccines for human use. So long as animal prod- 
ucts such as serum have to be used, the danger 
always exists of bringing in contaminating viruses 
which may be present in serum. Protein free 
media which support the growth of cells and the 
propagation of viruses have the same significance 
to virology, therefore, that sterile media in gen- 
eral have to bacteriology. Rhesus monkey kidney 
cells have already been adapted to chemically de- 
fined, protein free media, and an encouraging start 
has already been made (through 3 culture genera- 
tions) with "green monkey" kidney cells. 

Metabolic studies by Doctors Westfall, Evans, 
Sanford, and Bryant and their associates are be- 
ing continued with respect both to what cells take 
out of culture media and what they put back that 
was not there before, or was there in different 
quantity. These basic studies underlie a number 
of fundamental problems, such as (a) the fur- 
ther improvement of media (e.g., it was partly 
upon the basis of such studies that successful 
media were devised) ; (b) identification of meta- 
bolic pathways and intracellular mechanisms that 
may be involved in neoplastic transformation in 
vitro; (c) designing of counter measures which 
may aid in combating the neoplastic process; (d) 
understanding of normal life processes at the bio- 
chemical level. 

The appearance of extremely high amounts of 
the enzyme arginase in a clone derived from a 
cell strain which showed only barely detectable 
amounts of this enzyme was reported last year. 
Another clone showed a moderate increase in this 
enzyme. These findings have been found to be 
characteristic of the respective clones in further 
investigations, and sufficiently stable under con- 
stant tissue culture conditions to serve as chemical 
markers for a new type of study involving the 
possibility of transfer of genetic material between 
mammalian cells, i.e. "hybridization", in vitro. 
Such studies are now in progress in this and other 
laboratories who have been supplied with the 
characteristic clones. 

Other results of interest were the findings in 
collaboration with Dr. Kuff (Lab. of Biochem- 
istry) of a higher B-glucuronidase activity in 
freshly explanted mouse liver cells than in orig- 



inal liver from which the explants were derived. 
Another interesting aspect of this study was that 
the enzyme activity was considerably influenced 
by the medium used for cultivation of the cells. 
These alterations m enzyme activity illustrate 
the fact that "metabolic transformations" occur in 
tissue culture as well as certain other types of 
transformations that have long been known to oc- 
cur i.e., malignant transformation, and chromo- 
somal changes. 

2. Malignant Transfor3iation in Vitko. Doc- 
tors Sanford, Evans, Westfall, and Earle and 
their associates are continuing their studies of 
many years duration on the malignant transfor- 
mation of cells in vitro. Every long-term strain 
of C3H mouse cells established from normal tissue 
and maintained in the laboratory has in the past, 
when adequately tested by injection into mice of 
the strain of origin, demonstrated a capacity to 
give rise to malignant neoplasms. Such lines had 
at some time in their life been grown in media 
containing foreign proteins such as those of em- 
bryo extract and serum. It therefore seemed of 
importance to determine whether cell lines estab- 
lished and maintained in chemically defined, pro- 
tein free, media would also show such transfor- 
mation. Parallel lines of cells of the same origin 
were therefore set up in protein-free media and 
in media contaming sera. The cells grown in 
media containing serum showed ability to give 
rise to sarcomas after 124 days in vitro. On the 
other hand the cells in protein-free media have not 
yet shown such capacity, after growth for 21 
months iii vitro. It remains to be determined 
whether they will eventually imdergo malignant 
transformation or whether they can be continued 
indefinitely in protein-free media without under- 
going malignant change. Additional studies for 
this purpose are in progress. 

C. Instrumentation and Methods 

An important technical development during tlie 
past year has been the working out of a successful 
method by Doctors Evans and Bryant for freez- 
ing and storage of certain cell lines maintained in 
protein-free, chemically defined media. This is 
of importance to a large national program now 
engaged in efforts to characterize and standardize 
various lines of cells for use in basic studies on 



68 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



cancer, growtli of specific viruses, etc. The pro- 
cedure requires only the addition of glycerine, 
a chemically defined ingredient. Success is crit- 
ically related to speed of freezing and thawing, 
the concentration of glycerine as well as to the 
rate at which the glycerine is removed from the 
cells after thawing. Storage is at —180° C, in a 
liquid nitrogen refrigerator. 

Another significant development in the technical 
area has been the demonstration by Mr. McQuil- 
kin and Dr. Earle of the value and potentialities 
of the time-lapse cinemicrographic analysis of cell 
populations in vitro. The basic apparatus has 
been under test for several years and methods have 
now been worked out for the measurement of 
various cellular events, including cell growth, mi- 
gration, adliesiveness to glass substrate, timing 
of events in mitotic cycle, etc. A tremendous 
amount of time has been required to transform 
the basic data on film to measured parameters 
which can be tabulated and further analysed. 
Dr. Earle and Mr. McQuilkin are now looking 
into the possible utilization of modem electronic 
scanning devices which may be capable to trans- 
forming film data directly to coded information 
which can be handled by electronic computers. 
Such a possibility would make feasible the explor- 
ation and study of intracellular life processes not 
now approachable with visual and manual meas- 
urement and correlative procedures. 

Studies on the instrumentation and methods for 
growth of mammalian cells in large batch lots are 
being continued by Doctors Earle and Bryant. 
These include suspension cultures of various sizes 
as well as continuous-flow type of apparatus 
which permit exchange of culture fluids without 
disturbance of cells during continuous operation. 



in. Other Areas of Investigation 

(A) Carcinogenesis 

1. DrFPUSION-CHAMEER INDUCTION' OF PlASMA- 

Cell Tumoks : Dr. Merwin reported last year that 
plasma cell tumors had been induced in mice in 
which blank diffusion chambers (i.e., chambers 
containing no living tumor or other tissue) had 
been placed in their peritoneal cavities. This was 
a follow-up study of an earlier observation that 
such tumors arose in mice bearing intraperitoneal 



chambers containing various types of tumor and 
other tissues. 

Additional studies have been carried out to test : 
(1) blaixk chambers of different sizes ; (2) the sep- 
arate plexiglas disks, membranes, and other parts 
of which the chambers are composed; and (3) 
boriiigs of different sizes of the plexiglas compo- 
nent of the chambers. 

It was found that: (1) Large chambers induce 
more plasma-cell tumors than smaller ones; (2) 
Discs of the three chamber materials (i.e., plexi- 
glas, millipore filters, and the cement used in 
assembly) induced plasma cell tumors whereas 
small pieces of plexiglas in several forms do not ; 
(3) plexiglas borings of relatively larger size 
induced many plasma cell tumors however. A 
correlation was found between the induction of 
plasma-cell timiors and the development of fibrosis 
around the foreign materials. The factors asso- 
ciated with the induction of plasma-cell tumors 
by these materials appear to be similar to those 
reported by Oppenlieimer and others for the in- 
duction of sarcoma in rats by cellophane and other 
plastics. 

When a certain type of tumer tissue (a sarcoma 
of a BALB/c mouse) was placed inside the cham- 
bers the incidence of plasma cell tumors was in- 
creased considerably over that obtained with blank 
chambers alone. Another type of tumor tissue 
(isologous mammary tumor) placed within the 
chambers increased the incidence of sarcomas of 
the type occasionally induced by blank chambers. 

2. Ultrasteuctuee or Plasma-Cell Tumors : In 
collaboration with Doctors Merwin and Potter 
(Lab. of Biology), Dr. Dalton has carried out ex- 
tensive studies on the ultrastructure of plasma- 
cell tumors of mice as well as upon the human 
counterpart of such tumors, namely multiple 
myeloma. Comparative studies were also made 
on normal plasma cells and on 6 fibrosarcomas of 
mice. 

Normal plasma cells of the mouse were char- 
acterized by a wide range, from cell to cell, in size 
of the cisternae of the ergastoplasm. This wide 
range was not noted in neoplastic plasma cells. 
Neoplastic plasma cells of the mouse regularly 
contained doughnut shaped (Tj^pe A) virus-like 
particles in the cisternae of the ergastoplasm. 
The particles were found to originate by budding 



NATIONAL CANCER INSTITUTE 



69 



from tlie endoplasmic reticulum. They were not 
observed in normal plasma cells nor in multijale 
myeloma cells in man. The particles resemble the 
immature form of certain known viruses but they 
are not considered by Dr. Dalton to represent 
etiological agents responsible for the induction of 
plasma cell tumors of mice since extensive efforts 
by Dr. Merwin to transmit the neoplasm by meas- 
ures that have been successful with other murine 
tumor viruses failed. 

The malignant cells of multiple myeloma were 
observed to be similar to the cells of plasma cell 
tumors of mice in ultrastructure, except that they 
did not contain the characteristic "A" type 
particles. 

JSTo correlation could be established between 
variations in detail of fine structure of cells of 
plasma cell tumore of the mouse and the type of 
serum globulin produced. 

3. Transimission or Hamstek Tumoes bt Feeding 
OF Tumor Cells : A previous report by Brindley 
and Banfield ( J.N.C.I. 26 : 949, 1961) showed that 
a reticulum-cell sarcoma of the hamster could be 
transmitted to other hamsters by feeding. Sub- 
mucosal tumors appeared in the larynx and 
pharynx of recipients. Dr. Stewart has repeated 
these studies with three different transplantable 
tumors of hamsters wliich arose during the course 
of other investigations. Two of the three tumors 
were transmissible by the feeding of intact tumor 
cells. One of them, a leukemia, produced gen- 
eralized leukemia witliin 8 to 12 weeks after being 
fed. The other, a lymphosarcoma produced 
laryngeal tumors of the same type 4-8 weeks after 
being fed. A myxosarcoma showed no evidence 
of transmissibility on feeding. Tumors have thus 
far failed to appear in hamsters inoculated with 
cell-free filtrates of these tumors or with fluids 
from tissue culture explants of them. 

B. iTrmvunology and Serology 

Except for the polyoma virus, which differs in 
immunological and biological properties from 
other known tumor viruses of animals, the usual 
serological and immunological methods for 
diagnosis and quantitative assay of viruses of 
infectious diseases have not been readily appli- 
cable to the tiunor viruses. Dr. Fink has spent a 



considerable amount of effort during the past 
year m attempts to develop methods applicable 
to tmnor viruses, using the Rous sarcoma and 
Moloney leukemia viruses as laboratory models. 

Little success has beeen achieved thus far with 
the ]\Ioloney virus, but substantial progress has 
been made with the Kous sarcoma virus model. 
Using the "high potency" preparations of Rous 
sarcoma virus now available. Dr. Fhik has been 
able to prepare strong heterologous antisera 
against tliis virus in rabbits. The ability of the 
antisera to neutralize the virus persisted after ab- 
sorption of the serum with sheep erythrocytes (to 
remove antibody against Forssman antigen) and 
with normal chicken tissues (to remove antibody 
against normal cliicken antigens). This fuiding 
strongly supports the conclusions that the Rous 
sarcoma virus does not contain the Forssman 
antigen, or normal chicken antigens as essential 
components. The absorbed heterologous antisera 
also fixed complement specifically with Rous sar- 
coma virus, in a titer of 1 : 80. This acliievement 
now make possible the use of the complement 
fixation reaction for diagnosing and titrating the 
virus. 

It has long been known that antisera induced in 
chickens fail, in general, to fix complement, due 
to some pecularity of the chicken. Dr. Fink also 
failed to demonstrate complement fijsation with 
the Rous sarcoma virus when whole antisera of 
chickens were used. Such antisera were capable 
of neutralizing the biological activity of Rous 
sarcoma virus however. In the further investiga- 
tion of cliicken antisera, Dr. Fink has found that 
the gamma globulin fraction of chicken antiserum 
against the Rous sarcoma virus separated by am- 
monium sulfate precipitation, will fix complement 
wlule the whole antiserum will not. It is probable 
that this successful accomplishment with homol- 
ogous sera will permit the development of comple- 
ment fixation tests which can be used for detecting 
antibody in epidemology type studies in chickens. 
In other studies on methodology, Doctors 
Zeigel, Fink and Kuff (Lab. of Biochemistry) are 
collaborating in attempts to apply the teclmique 
of conjugating antibody with ferritin so tliat it 
can be visualized under the electron microscope. 
Examinations of such ferritin labeled antibodies 



70 



ANNUAL REVIEW OF rNTRAMTJRAL RESEARCH 



under the electron microscope have revealed com- 
plexes not seen with ferritin control preparations, 
indicating that conjugation, as reported by others, 
may have been successfully achieved. Serological 
studies to confirm this are in progress. 

C. Radiation 

In continuation of studies on tissue growth by 
means of light microscopy through the trans- 
parent chambers developed by Algire, Dr. Merwin 
has determined the influence of proliferating nor- 
mal endothelial cells on the growth of x-irradi- 
ated transplants of tmnor tissue and of the healing 
of wounds in irradiated areas within transparent 
chambers. Previous studies had shown that ir- 



radiated tiunor failed to grow, and that healing 
was delayed m irradiated areas. 

It was found that scattered foci of endothelium 
still capable of growing were present within ir- 
radiated tumors. Such growing endothelium sup- 
ported the growth of surrounding tumor at a 
normal rate, but there was no growth of capillaries 
into adjacent areas which had become anoxic due 
to inadequate vascular supply. The introduction 
of normal, unirradiated endothelium into irra- 
diated areas of wounds hastened the healing proc- 
ess. Further studies are necessary to determine 
whether the introduction of normal endothelium 
would be effective in the treatment of wounds, 
particularly surgical wounds, in irradiated areas. 



NATIONAL HEART INSTITUTE 



INTRODUCTION 

As in the past, this review of intramural re- 
search in the Heart Institute consists of the as- 
sembled reports of tlie leaders of its major researcli 
groups. It will be apparent to the reader that it 
describes work of considerable breadtli, depth and 
variety. We hope that it will also be apparent 
that is describes work of considerable scientific 
merit. 

Note should be made of certain organizational 
changes which are reflected in this report and 
others which will be involved only in the next. 
This year for the fiirst time the group in Experi- 
mental Therapeutics, Clinical Endocrinology and 
Cardiology report as separate branches. This in 
reality constitutes recognition of an autonomy 
under which these groups have operated for sev- 
eral years. In the coming year it is anticipated 
that the Laboratory of Chemistry of Natural 
Products, the head of which left the NIH this 
year, will be abolished. A segment of its f imctions 
will be contmued by a section to be incorporated 
into another laboratory. The impending de- 
parture of the head of the Laboratory of Cellular 
Physiology and Metabolism will be a serious loss 
to the scientific leadership of the Institute. The 
solution to the problem it creates has not yet been 
determined. However, it is anticipated that the 
Sections on Eniiymes and Metabolism will be es- 
tablished as separate laboratories in recognition of 
the stature and independence of these groups. 

LABORATORY OF CELLULAR PHYSIOLOGY 
AND METABOLISM 

Section on Cellular Physiology 

The program of the Laboratory of Cellular 
Physiology and Metabolism, Section on Cellular 
Physiology, has contmued in the direction of 
investigating the structure, synthesis, fiuiction 
and genetic control of protein molecules. Experi- 



mental results over the past year have reinforced 
earlier conclusions that the three-dimensional 
structure of proteins is derived directly from 
their primary amino acid sequence. Thus globu- 
lar molecules, previously converted to random 
chains in solution by reduction of disulfied 
bridges, are regenerated to the native enzyme 
upon reoxidation of the SH groups in spite of the 
extremely improbably occurrence of correct pair- 
ing of the SH gi-oups in disulfide linkage. With 
the enzyme ribonuclease, for example, 105 forms 
of the protein, containing 4 SS bridges, are pos- 
sible when its 8 half -cystine residues are oxidized 
in a random fashion. Nevertheless, theoreticallj' 
maximmn yields of native enzyme can be obtained 
by a spontaneous reaction. 

In addition to studies specifically directed at 
protein molecules themselves, a number of mves- 
tigations, reported below, have been carried out 
on more biological aspects of biochemistry, includ- 
ing such processes as electron transport in meta- 
bolic systems, the biochemistry and cytology of 
cell transport, and the mechanisms involved in 
the transport of fat. 

Optimum conditions have been worked out for 
the formation of native ribonuclease from the re- 
duced molecule containing 8 SH groups. These 
conditions, involving the use of low protein con- 
centrations at a pH of 8.0 lead reproducibly to 
full regeneration of activity. The kmetics of the 
reoxidation process have been examined in detail 
and an interesting lag period has been observed 
during which no activity appears in spite of con- 
siderable disulfide bridge formation and the 
assimiption of complex internal structure. Pre- 
liminary experiments suggest that absence of 
activity is due to the formation of randomly 
paired SH groups which then, subsequently, re- 
arrange to yield the proper set of i disulfide 
bridges. Further evidence for this rearrange- 
ment hypothesis was obtained from experiments 
in which fornas of ribonuclease, deliberately pre- 

71 



72 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



pared with random disulfide bridging, were ex- 
posed to dilute solutions of various sulfhydryl 
compoimds. Such compounds catalyzed a re- 
arrangement and led to high yields of the native 
configuration (which must therefore be thermo- 
dynamically the most probable form) . 

The general hypothesis that the primary se- 
quence alone determines tertiary structure has 
been examined using other proteins as models. 
By methods similar to those used with ribonu- 
clease, it has been shown that egg white lysozyme 
can also be subjected to reduction-reoxidation 
with efficient regeneration of activity. Experi- 
ments with trypsin were initially unsuccessful be- 
cause of the extreme soliibility of the reduced 
form. Subsequently, the molecule has been at- 
tached to carboxymethylcellulose and the result- 
ing insoluble derivative has been used to prepare 
columns. The trypsin in these columns could be 
reduced and reoxidized with approximately 
5-10% regeneration of enzymatic activity. Fur- 
ther tests on other protein molecules are underway. 

In a continuation of studies of the role of vari- 
ous side chain interactions in the reoxidation 
process, ribonuclease has been reduced and reoxi- 
dized following the attachment of polypeptide 
chains to the epsilon amino groups of lysine resi- 
dues. These derivatives, containing as many as 
70 added alanine residues, can still be successfully 
reoxidized after reduction. The amino groups 
themselves must, therefore, not be directly re- 
quired as information for refolding but may, per- 
haps, contribute only to the total charge of the 
protein in solution. This conclusion is reasonable 
since the added polyalanine side chains also pos- 
sess a single terminal positive charge. On re- 
moving positive charges by acylation, the capacity 
to refold is lost. 

These studies with polypeptide derivates are of 
added interest since it has beeen further noted 
that 3 of the 11 free amino groups in ribonuclease 
are completely resistant to peptidylation and are 
probably hidden within the structure of the pro- 
tein. The results suggest that such methods 
might be useful in studying the chemical topog- 
raphy of poteins in general, and it is planned to 
apply these techniques to several other test 
proteins. 

The ready formation of native ribonuclease 
from the reduced, random chain has suggested 



the possibility that one might attack the total 
synthesis of ribonuclease with some chance of suc- 
cess. Before beginning such studies, certain 
questionable features of the sequence, as reported 
by investigators at the Eockefeller Institute and 
in this Laboratory, were reinvestigated. The 
sequence in question has been corrected and the 
results from both laboratories now agree in full. 
The primary structure of the protein now appears 
to be correct. 

The plan for chemical synthesis of the ribonu- 
clease chain involves the limited cleavage of the 
polypeptide chain into 2 to 5 fragments, using 
trypsin to cleave, specifically, the peptide bonds 
involving arginine. Trypsin is chosen since it is 
the most specific of the known proteolytic enzymes. 
To limit cleavage to arginyl bonds, it is necessary 
to block the epsilon amino groups of lysine since 
peptide bonds involving this amino acid are also 
cleaved by trypsin. The blocking group must be 
easily removable to permit the ultimate regenera- 
tion of native enzyme. Tlie reagent chosen for 
this purpose was the ethylthiolester of trifluoro- 
acetic acid, and it has been shown that, using this 
reagent, all the requirements listed above can be 
achieved. A critical step in the acylation and 
deacylation involves the "correction" of incori'ect 
disulfide bridges that have been formed by disul- 
fide interchange during the acylation reaction. 
The correct pairing of SH groups was achieved by 
complete reduction, followed by oxidation of the 
reduced chain under optimal conditions. 

When samples of trifluoroacetyl ENase were re- 
duced, the resulting material yielded 5 fragments 
upon trypsin digestion according to prediction. 
The problem of joining these fragments together 
involves the formation of peptide bonds between 
each of the fragments arranged in their proper 
order. Since ribonuclease contains a niunber of 
free carboxyl groups which would also react dur- 
ing the chemical formation of peptide bonds, these 
must also be blocked in a reversible way before 
the reconstruction experiments can be initiated. 
Studies are now underway to investigate the suit- 
ability of esterification of these groups with 
methanol. 

Studies on the structure of egg white lysozyme 
have been continued, and the total elucidation of 
the sequence is nearing completion. The deter- 
mination of the disulfide bridges will be under- 



NATIONAL HEART INSTITUTE 



73 



taken in the next few months. As an additional 
point of interest regarding this enzyme, the pro- 
tein has been isolated from incubations of minced 
hen's oviduct to -which tritiated leucine had been 
added. The radioactive protein was isolated in 
pure form and leucine containing peptides were 
isolated from enzymatic digests of the polypep- 
tide chain. The specific radioactivity of the in- 
dividual leucine residues differed from one an- 
other by as much as 250% and the specific activity 
was f omid to increase in the order in which these 
residues appear along the chain. Thus leucines 
near the amino terminal end were much less radio- 
active than those near the carboxyl terminal end. 
These observations were consistent with a model 
which depicts protein biosynthesis as a process of 
unidirectional growth of a polypeptide chain 
(along a template) beginning at the amino and 
terminating at the carboxyl end of the chain. 

The previously postulated involvement of lipid 
complexes or lipid compoimds of amino acids in 
protein synthesis has been further exammed, both 
in minced oviduct tissue and in cells of E. coli. 
It has been found that one particular purified 
amino acid lipid complex involves a covalent bond 
sensitive to hydroxylamine between the amino acid 
and the lipid moiety. This material was shown 
to undergo rapid metabolic turnover at a rate con- 
sistent with involvement in protein biosynthesis. 
Some evidence was also obtained for similar com- 
plexes in the E. coli system. The characteriza- 
tion of these materials, as derived from bacterial 
cells, has been much more difficult and requires 
further work. The overall findings have been 
included in a model suggested as a consistent inter- 
pretation of available observations on protein 
synthesis. 

Studies on the genetic control of the structure 
of a lysozyme isolated from bacteriophage parti- 
cles have been continued. A "map" of a number 
of mutational sites that lead to modifications in 
the functional aspects of this enzyme has been 
constructed and the sequence of the enzyme isolated 
from the parent strain of phage is well on its way 
to completion. It is hoped, during the coming 
year, to complete this latter investigation and to 
compare the structures of "mutant" lysozymes 
with that isolated from the parental strain. The 
results should give a direct answer to the question 
of the colinearity of the genetic map and the pri- 



mary sequence and should, furthermore, provide 
direct information on the nature of the genetic 
code relating DNA structure with protein 
structure. 

Studies on the structure and function of muscle 
proteins involved in contraction are continuing. 
Tlie major component of the contractile system of 
muscle, myosin, has been investigated with regard 
to its structure, and the relationships between cer- 
tain aspects of structure and ATPase activity have 
been examined. Physical-biochemical studies 
have shown that the long myosin molecule is com- 
posed of three identical polypeptide chains as- 
sociated together in the form of a three-stranded 
rope. Tliis complex may be dissociated into single 
strands and re-formed by careful removal of the 
dissociating agent. ATPase activity was, how- 
ever, not regenerated, and experiments to over- 
come this deficiency are in progress. Other 
experiments have involved the blocking of certain 
essential SH groups in myosin that are known 
to be required for enzymatic activity. By enzy- 
matic digestion of the blocked protein, followed 
by peptide separations, fragments have been iso- 
lated, in pure form, containing the blocked group. 
The determination of their structures should fur- 
nish direct information on the chemistry of the 
active center of myosin- ATPase. Similar studies 
on a second essential component of the contractile 
system, actin, have been initiated. 

A new project in the Laboratory is concerned 
with the biochemistry and cytology of cell trans- 
port. A convenient mutant of the slime mold, 
Dicytiostelium discoideum, which does not aggre- 
gate but which grows vigorously in the amoeboid 
form, has been analyzed extensively for its phos- 
pholipid components. Experiments are now un- 
derway to prepare cell walls from these organisms 
and to examine their structure by chemical and 
electron microscopic methods. It is further 
planned to study the metabolic events occurring in 
cells during the phagocytosis and uptake of vari- 
ous materials. The uptake and transport of fat, 
in particular, will be investigated in these cells. 
Concurrently, studies on the transport of fats are 
beinff pursued with the lactating mammary glands 
of guinea pigs as an active fat metabolizing organ. 
Attempts will be made to correlate the ability of 
the gland to assimilate triglycerides with the lipo- 
protein lipase activity of the tissue. 



74 



ANISTJAL REVIEW OF INTRAMURAL RESEARCH 



The nature of the reaction catalyzed by lipo- 
protein lipase as well as the chemistry of the 
enzyme itself are under continuing investigation. 
In particular, chemical studies are in progress on 
the inhibition of the enzyme by polyanions and 
polycations. It is hoped that the study of such 
inhibitors will throw some light on the mechanism 
of the hydrolysis catalyzed by this specific lipase. 

Section on Metabolism 

Wliile it is clearly recognized that the patho- 
genesis of atherosclerosis is complex and multi- 
f acted, there is adequate evidence to show that ele- 
vated levels of blood lipids play some role in the 
development of this important disease. A great 
deal of research has been done on the factors con- 
trolling levels of blood lipids but much of it is 
purely descriptive and empirical. Before the 
problem of hyperlipidemia can be approached 
rationally we shall have to acquire deeper insights 
into the mechanisms controlling the production 
and degradation of the serum lipoproteins and 
their component parts. Investigators in this sec- 
tion continue to concentrate their efforts primarily 
on basic stiidies of lipid metabolism directed ulti- 
mately at elucidation of the complex metabolic 
pattern that determines steady state concentra- 
tions of serum lipids. 

Pathroay and Inhibitors of Cliolesterol Bio- 
synthesis 

Previous work in this laboratory has established 
that reduction in the rate of hepatic synthesis of 
cholesterol can lead to a depression of serum 
cholesterol levels both in animals and in man. A 
variety of inhibitory agents has been exxjlored in 
recent years. Two years ago we established the 
mechanism of action of triparanol (MEE-29), 
showing that it blocked the last step in cholesterol 
biosynthesis — the reduction of desmosterol to 
cholesterol. The clinical effects of the drug have 
been disappointing. Cholesterol levels are de- 
pressed very considerably (on the average 30 to 
35%) but desmosterol accumulation is so great that 
total serum sterols are only reduced by about 15%. 
Some investigators have held out the hope that 
desmosterol might prove to be less atherogenic 
than cholesterol but studies in this laboratory have 
now shown that desmosterol is laid down in 



normal aorta and in atheromata of rabbits at a 
rate indistinguishable from that at wliich choles- 
terol is laid down. 

The studies on the mechanism of action of tri- 
paranol have opened several interesting lines of in- 
vestigation. These studies provided important 
evidence that desmosterol is a normal intennediate 
in cholesterol biosynthesis. Clinical studies com- 
pleted this year show that desmostei'ol is an excel- 
lent precursor for bile acid synthesis and for 
adrenal steroid synthesis. The possibility that 
the introduction of a double bond into the side 
chain occurs during the conversion of cholesterol 
to bile acids must now be considered. 

Application of the powerful teclinique of thin- 
layer chromatography has made it possible to 
study more simply the reductive reaction blocked 
by triparanol. Preliminary results show that the 
drug inhibits reduction of lanosterol to dihydro- 
lanosterol, suggesting that perhaps the same 
enzyme is involved in reduction of the side chain 
double bond whatever the structure of the sterol 
nucleus. Tliis enzyme has been shown to require 
TPNH as cofactor and to depend upon a reduced 
sulhydryl group for its activity. An unidentified 
sterol component of high specific radioactivity has 
been isolated in the course of these studies and 
its chemical structure is currently under 
investigation. 

The Heart Institute was fortunate in having 
Dr. George Popjak as a Visiting Scientist during a 
part of the past year. He collaborated in studies 
which have established the details of the mecha- 
nism of squalene formation. It was shown that 
two molecules of f amesyl pyrophosphate condense 
in an asymmetric fashion to yield squalene. One 
atom of hydrogen from one of these two molecules 
is replaced by hydrogen derived from TPNH. 
This unusual finding, which corrects previously 
reported studies done in other laboratories, may 
disclose a new type of condensation reaction in 
mammalian systems. 

Metabolism of Cholesterol Esters 

The fatty acids associated with the cholesterol 
ester fraction in the serum of animals and of man 
are higlily unsaturated. This has suggested the 
possibility that the well-known effects of unsatu- 
rated fats in the diet on serum cholesterol levels 
may be due to changes in cholesterol ester metab- 



NATIONAL HEART INSTITUTE 



75 



olism. In order to explore this possibility more 
basic information about the metabolism of choles- 
terol esters is required. Studies initiated this year 
were designed to explore several aspects of the 
problem. The esterification of cholesterol in the 
intestinal wall during absorption has been studied 
and preliminary results fail to reveal any differ- 
ences in the rate at which saturated and unsatu- 
rated fatty acids are esterified. 

The greatest fraction of newly absorbed choles- 
terol is known to appear in the chylomicrons in 
the form of cholesterol esters. The fate of these 
cholesterol esters has been studied by intravenous 
injection of chylomicrons containing C^'^-labeled 
esters, obtained by the collection of chyle from the 
thoracic duct of rats previously fed C^*-choles- 
terol. Radioactive free cholesterol was largely 
removed from these preparations by an exchange 
process. It was shown that more than 80% of 
the labeled ester cholesterol of the injected 
chylomicrons was taken up by the liver in 20 
minutes. Over the next 24 hours radioactivity 
appeared slowly in the blood and other tissues in 
free form. Thus it appears that ingested choles- 
terol is esterified during its passage from the in- 
testinal lumen into the chyle, is deposited in ester 
form in the liver very rapidly, and then imdergoes 
hydrolysis in the liver to be released as free 
cholesterol once again. 

The nature of the enzyme systems that hydro- 
lyze and synthesize cholesterol esters in the liver 
is under investigation. The cholesterol esters 
present in the soluble fraction have been purified 
30 to 40-fold. The nature of this enzyme and its 
range of substrate specificity is being explored. 

Effects of Dietary Fat on Cholesterol Metabolism 

This project, being carried out in collaboration 
with Dr. Sven Lindstedt of the Karolinska In- 
stitute in Stockholm, has been continued in order 
to clarify results previously obtained in the study 
of bile acid metabolism. Results analyzed to date 
show that there is a consistent but small effect of 
unsaturated fats on bile acid formation. When 
all other factors are held constant the substitution 
of imsaturated fats for saturated fats causes a 
slight increase in bile acid excretion. An impor- 
tant outcome of these studies is the demonstration 
that the pattern of fecal bile acids is considerably 
altered as a function of the kind of dietary fat 
offered. Consequently the determination of the 



fecal excretion of any single bile acid component 
will not give a true picture of the total fecal bile 
acid excretion. Most of the results reported by 
other laboratories have been based on measure- 
ments of only one or tAvo bile acid components. 
While quantitative evaluation of our results is not 
yet complete it appears that the observed changes 
in bile acid excretion are not large enough to 
account for the effects of dietary fats on seiaun 
cholesterol levels. 

Lipidoses and Hyperlipemia 

A new clinical entity, characterized by abnormal 
storage of cholesterol esters in the tissues, has been 
discovered. Two siblings living on the island of 
Tangier have been studied in great detail. These 
patients appear to have a complete deficiency of 
tti-lipoprotein or high-density lipoprotein (HDL) . 
Although they lack any demonstrable HDL they 
are able to form chylomicrons. This finding 
forces revision of theories implicating HDL pro- 
tein as an integral part of the chylomicrons. The 
j8-lipoprotein level in these patients is not particu- 
larly abnormal and it appears therefore that the 
production of these two density classes of lipopro- 
tein is separately and specifically controlled. 

An extensive survey of the population on the 
island of Tangier is being carried out. A very 
high percentage of the population shows a marked 
reduction in HDL concentration. An analysis of 
the genetic relations involved suggests that the 
observed abnormality is not due to a single allelic 
abnormality and nongenetic factors have not been 
entirely ruled out. However, it seems most likely 
that this syndrome is a genetically determined de- 
rangement of lipoprotein metabolism. 

Another patient, apparently unrelated to those 
with Tangier disease, has been discovered with 
marked hypercholesterolemia, hepatic enlarge- 
ment, and striking elevation of cholesterol ester 
concentration in the liver. Again, this patient 
shows a marked decrease in HDL. "\^1iereas pa- 
tients with Tangier disease have veiy little im- 
munochemically identifiable HDL, this patient, 
even though she has veiy little lipoprotein NA^ith the 
density of normal HDL, does have immunochemi- 
cally demonstrable HDL-protein in her serum. 

Extensive studies of the lipid metabolism in 
these interesting syndromes are underway. The 
results should be of great value in detennining the 
normal function of higli density lipoproteins. 



76 



ANTSrUAL REVIEW OF INTRAMURAL RESEARCH 



It as been recognized for some time that the 
clinical syndrome of hyperlipemia is heterogene- 
ous but no systematic classification has been possi- 
ble. An extensive survey is underway in an 
attempt to differentiate hyperlipemias of different 
metabolic origins. The use of a more sensitive 
teclinique for measuz-ement of lij)olytic activity 
after injection of heparin has revealed that pa- 
tients with fat-induced hyperlipemia have, as a 
group, a diminished response. Siblmgs and par- 
ents of patients with hyperlipemia also tend to 
have lower values than normal even though they 
may not exhibit hyperlipemia. The enzyme assay 
method has been made available to a number of 
other clinics in the hope that the application of a 
standardized technique to large groups of patients 
may clarify this syndrome further. At least one 
other type of hyperlipemia can now be clearly 
separated — carbohydrate-induced hyperlipemia. 
In contract to the patients just discussed, who 
develop hyperlipemia on ingestion of fat, the car- 
bohydrate-induced lipemics develop hyperlipemia 
only on diets high in carbohydrate. These patients 
show normal levels of clearing activity after in- 
jection of heparin. 

Xanthomatous tissue removed from a patient 
with xanthoma disseminatum (a cutaneous form 
of Hand-SchuUer-Cliristian disease in which 
plasma lipids are normal) synthesized cholesterol 
from acetate at a rate five times that of normal 
skin while eruptive xanthomas from a patient with 
hyperlipidemia had an essentially normal rate of 
synthesis. This finding, although it requires con- 
firmation in a few more patients, is the first bio- 
chemical proof of an earlier theory of Thann- 
hauser that the lesions in the former disease were 
probably synthesizing most of the lipid stored 
within them. 

Metabolism of Adipose Tissue and Hormonal Ef- 
fects on Fat Mohilisation. 

This laboratory is continuing with a broad series 
of investigations on the biochemistry of adipose 
tissue. It is considered essential to obtain a deeper 
insight into the factors controlling the metabolism 
of adipose tissue since without this infonnation 
it wiU not be possible to reach understanding of 
the complex hormonal influences on this tissue. 

In previously reported studies the step by step 
mechanism of triglyceride synthesis in adipose 



tissue has been determined and during the past 
year some of the steps involved have been charac- 
terized in greater detail. Alpha-glycerophosphate 
has been shown to be an obligatoiy acceptor of 
fatty acids for triglyceride synthesis in this tissue. 
More sensitive teclniiques still fail to demonstrate 
the presence of any enzymes that can phosphory- 
late free glycerol. It has been shown that a nimi- 
ber of glycolytic intermediates can give rise to 
""glycerophosphate by well laiown reactions. 
Evidence has been obtained that adipose tissue 
has transaldolase activity but the physiologic im- 
portance of this reaction in adipose tissue is still 
uncertain. The tissue contains an active phos- 
phatase but this enzyme has activity only at an 
acid pH. It will be interesting to determine 
whether changes in the activity of this enzyme 
may influence rates of triglyceride synthesis. It 
has also been shown that a-glycerophosphate can 
be formed by a transphosphorylation reaction and 
again further studies will be needed to determine 
the physiological importance of this reaction. 

The last step in triglyceride synthesis in adipose 
tissue has been studied in further detail. A par- 
ticulate enzyme system fi'om chicken adipose tis- 
sue cataylzes the reaction of diglycerides with 
activated fatty acids to form triglycerides. Di- 
glycerides containing at least one misaturated 
fatty acid were more reactive than saturated di- 
glycerides. However, the relative positions of 
the saturated and unsaturated fatty acids in mixed 
diglycerides did not appear to influence the rate 
of this reaction very significantly. 

Because glycerol can not be activated and re- 
utilized for triglyceride synthesis the rate of re- 
lease of glycerol has been used as a measure of 
the rate of a lipolysis independent of changes in 
the rate of triglyceride synthesis. Using this 
technique it has been shown that there is continu- 
ing lipolysis in adipose tissue under all conditions. 
Even when triglyceride synthesis is strongly stim- 
ulated by the addition of glucose, glycerol con- 
tinues to be released at rates not significantly dif- 
ferent from those seen under control conditions. 
This confirms earlier conclusions based on meas- 
urements of l-C^*-palmitic acid incorporation to 
the effect that glucose exerts its suppressive effect 
on fatty acid release primarily if not exclusively 
by stimulating triglyceride synthesis. Epineph- 
rine, norepinephrine, glucagon and adrenocortico- 



NATIONAL HEART INSTITUTE 



77 



tropic hormone all increase the rate of glycerol 
release, indicating that they stimulate the rate of 
breakdown of triglycerides. These findings are 
in conflict with tentative conclusions previously 
drawn on the basis of measurements of incorpora- 
tion of labeled fatty acids. In these earlier studies 
it was shovfn that the rate of incorporation of 
fatty acids into triglycerides was inhibited by 
these several hormones. More recent studies have 
shown that the pool of free fatty acids in the adi- 
pose tissue is heterogeneous. After incubation of 
the fat pad with labeled palmitic acid it has been 
shown that the free fatty acids in different frac- 
tions of the tissue can, under some circumstances, 
have widely different specific radioactivities. 
"Which of these fractions, if any, represents the 
true precursor pool remains to be determined. 
Preliminary studies, carried out in collaboration 
with Dr. A. T. James in London, appear to rule 
out a direct incorporation of free fatty acids from 
the medium and suggest that the figures obtained 
for triglyceride turnover in earlier experiments 
will not require very much revision. Further 
quantitative studies along these lines are planned. 
The nature of the lipolytic system in adipose 
tissue is being investigated since this appears to 
play a central role in controlling the mobilization 
of fatty acids. The properties of the enzyme sys- 
tem under investigation are quite different from 
those attributed to lipoprotein lipase. It has been 
shown that the activity of this enzyme system in 
tissues that have been exposed to epinephrine is 
increased, although the changes are not as large 
as the changes in the rates of release of fatty acids 
induced by epinephrine. The fact that the largest 
fraction of the enzyme becomes closely associated 
with the adipose tissue neutral fat and is con- 
centrated at the top of the tube when homogenates 
are centrifuged holds out hope that purification 
may be accomplished with relative ease. 

Metabolic Fate of Free Fatty Acids 

Previous studies in this laboratory showed that 
when free fatty acids were mobilized from adi- 
pose tissue at a high rate under the influence of 
catechol amines there was a marked net deposition 
of triglyceride in the liver and, to a lesser extent, 
in the muscle. In vitro studies have now shown 
that the rate of triglyceride synthesis in liver slices 
increases as a function of the concentration of 



fatty acids in the medium. The rate of fatty acid 
oxidation and the rate of phospholipid synthesis 
were increased but to a lesser extent. In vitro 
studies of skeletal muscle have also shown that 
the available concentration of free fatty acids in- 
fluences rates of triglyceride synthesis and rates 
of fatty acid oxidation in a similar fashion. 

It is now possible to synthesize the results ob- 
tained over the past several years into the follow- 
ing hypothesis concerning the fate of free fatty 
acids : 

The rate of mobilization of free fatty acids 
from adipose tissue is increased in the fasting 
state and under conditions of stress. Catechol 
amines, released directly at nerve endings or 
carried to the adipose tissue by way of the 
blood stream, strongly stimidate fatty acid 
release. During active fat mobilization the 
serum levels of FFA are elevated. This 
causes an increase in the uptake of FFA in 
the liver and a large part of the fatty acid 
taken up is converted to triglycerides there. 
If the rate of fat mobilization is very high 
these triglycerides accumulate in the liver. 
A part of the fatty acid brought to the liver 
is incorporated into lipoproteins and secreted 
back into the serum compartment. It is sug- 
gested that the elevation of serum lipopro- 
teins that is seen after injection of catechol 
amines or under stressful conditions is di- 
rectly or indirectly attributable to the extra 
load of FFA delivered to the liver. Tlie in 
vitro studies of skeletal muscle suggest that 
the rate of peripheral utilization of FFA is 
to some extent conditioned by the concentra- 
tions of FFA in the serum. It will be impor- 
tant to explore the possible hormonal control 
over this latter process. 
Previous studies in this laboratory established 
the remarkably rapid rate of turnover of serum 
free fatty acids. Kinetic analysis of these results 
was complicated by the unusual pattern at later 
time intei'vals. These activities have now been 
clarified by the finding that even highly purified 
radioactive fatty acids contain sufficient impurity 
to affect significantly the late portions of the serum 
disappearance curves. Only by purifying the 
labeled substrates by means of gas-liquid chroma- 
tography is it possible to obtain materials suiH- 
ciently pure to give unambiguous results. Con- 



78 



ANKTJAL REVIEW OF INTRAMURAL RESEARCH 



flicting findings reported from several laboratories 
are probably resolved and explained on the basis 
of contaminant radioactive materials in the prep- 
arations used. 

Using labeled fatty acids prepared by gas-liquid 
chromatography it has been shown that the un- 
saturated fatty acids (oleic and linoleic acids) 
disappear from the plasma FFA pool more 
rapidly than does palmitic acid. In view of the 
well recognized effects of dietary fats of different 
degrees of saturation on serum lipoprotein levels 
this finding may be of gi-eat significance. A num- 
ber of isotopically labeled fatty acids have been 
synthesized and exhaustively purified to permit 
extension of these studies. 

On Phospholipid Metabolism 

It has been postulated that the transport of ions 
through membranes may be intimately linked to 
phospholipid metabolism. Preparatory to in- 
vestigating this problem studies have been initiated 
to determine the properties of the system in red 
blood cells that synthesizes phospholipids. It has 
been shown that the ghosts (membranes) of 
human red blood cells can incorporate labeled 
fatty acids into phospholipids. Coenzyme A and 
ATP are both essential requirements but a-glyc- 
erophosphate does not stimulate incorporation. 
Since, in all other phospholipid-synthesizing sys- 
tems, a-glycerophosphate has been shown to be an 
obligatory intermediate, these findings suggested 
that the incorporation observed did not represent 
net synthesis. It has been shown that C^*-palmitic 
acid is incorporated primarily into lecithin and 
that the palmitic acid is located selectively in the 
alpha position. In other words it appears that 
there is an active turnover of preformed phospho- 
lipid molecules in the membrane. The possible 
correlation of this with the function of the mem- 
brane is the subject of further study. 

Metabolism of Ricinoleic Acid and Other Hydroxy 
Fatty Acids 

Castor oil is one of the most widely used agents 
in medicine and yet little or nothing is known 
about its mechanism of action. Ricinoleic acid 
makes up over 80% of the total fatty acid content 
of castor oil and it is almost certain that the im- 
usual effects of castor oil are related to this 
hydroxy fatty acid. It has been shown that the 



fatty acids of castor oil are absoi-bed both by the 
rat and by man. Eats maintained on daily intake 
of castor oil accimiulate significant amounts of 
ricinoleic acid in their adipose tissue. Castor oil 
undergoes enzymatic hydrolysis at rates compara- 
ble to those seen for the more usual oils but the 
rate at which ricinoleic acid is activated to form 
the coenzyme A derivative is much lower than that 
seen for the common fatty acids. Thus, it is pos- 
sible that free ricinoleic acid may accumulate and 
lead to purgation. Whether this is due to specific 
properties of ricinoleic acid or only to the fact 
that it accumulates in higher concentration re- 
mains to be determined. 

In vitro studies with rat adipose tissue have 
shown that hydroxy fatty acids (ricinoleic acid, 
9-hydroxystearic acid and 10-hydroxystearic acid) 
are incorporated into triglycerides but at rates 
significantly lower than the rate of incorporation 
of palmitic acid. At high concentrations in the 
medium these hydroxy acids inhibit the incorpora- 
tion of palmitic acid. 

In connection with clinical studies of the fate 
of ingested castor oil a technique for measurement 
of fecal fat had to be developed. Methods cur- 
rently in use failed to measure hydroxy acids be- 
cause these acids are so much more polar than the 
common fatty acids. A sample technique for 
quantitative collection and sampling of feces was 
developed and the method for extraction and 
determination of lipids was devised. 

Protein Metabolism 

The syndrome in which serum proteins "leak"' 
into the gastrointestinal tract and, because of pi'O- 
teolysis, are lost to the body (protein-losing gas- 
troenteropathy) has been studied in a wide variety 
of clinical conditions. The syndrome is seen in 
patients with a variety of gastrointestinal diseases 
recognizable by other clinical manifestations. In 
addition, however, it has been shown to occur in 
patients without clinically apparent gastro- 
intestinal disease. Now it has been shown 
further that patients with congestive heart failure, 
particularly those with constrictive pericarditis, 
may exhibit the sjaidrome. 

A new teclmique has been devised for demon- 
strating protein-losing gastroenteropathy. Al- 
bumin labeled with chromium'^ is injected intra- 
venously and the amoiuit of labeled chromium 



NATIONAL HEART INSTITUTE 



79 



appearing in the feces is measured. After tlie 
tagged albumin is lost into the intestine the chro- 
mium appears not to be reabsorbable and the 
recovery of it in the feces reflects the amount of 
protein leaking into the intestine. 

Basic studies on the structure of proteins of bio- 
logical interest have been carried out along two 
major lines. The structure of the fibrinogen 
molecule has been further investigated and it has 
been shown that trypsin destroys the ability of 
fibrinogen to form a clot, probably by splitting a 
single peptide bond. Wlien digestion is allowed to 
proceed further a large number of peptide bonds 
is split and the molecule appears to be reduced to 
three fragments of approximately equal size and 
shape. The relation of these findings to clot 
formation is the subject of further investigation. 

In the course of studies on serum albumin 
metabolism it was noted that the commonly used 
HABA dye method gives different color yields 
with the albumin of different species. These 
differences reflect the fact that the dye-albumin 
complex formed has different structures in differ- 
ent species. A systematic study of serum albumin 
from 10 species has been carried out. It has been 
shown that the albumins from closely related 
species form similar complexes while those of 
phylogenetically divergent species form distinct 
complexes. It is hoped that close analysis of the 
spectral characteristics of the dye-albumin com- 
plex may be useful in inferring structural char- 
acteristics of these different albumin molecules. 

Section on Enzymes 

The research program of the Section on En- 
zymes is concerned with studies of diverse bio- 
chemical reactions involved in the intermediary 
metabolism of one, two, and three carbon com- 
pounds and in the metabolism of lipids, amino 
acids, heterocyclic compounds and onium com- 
pounds. Led by the consideration that all living 
organisms make use of similar if not identical 
enzymatic reactions in the course of their metabo- 
lism, the biological material used for these studies 
consists mostly of specialized microorganisms 
that have been isolated on selected substrates and 
offer operational advantages over other experi- 
mental media for the specific studies undertaken. 
The various metabolic processes chosen for in- 



vestigation represent model systems that present 
unique opportunities to obtain an insight into bio- 
chemical mechanisms of general, fundamental 
significance. The results of these investigations 
are summarized below. 

One-Carbon Metabolism 

Formate Activation". Experimental evidence for 
the occurrence of formyl-CoA as an enzymatically 
active one carbon derivative has been obtained 
tlorough studies with cell-free extracts of G. 
hluyveri. Extending the studies of Lieberman 
and Barker who observed that extracts of this 
organism catalyze the formate-dependent de- 
composition of acetyl-phosphate, it has now been 
established that formyl-CoA is an intermediate 
m the overall process. Three coupled enzymatic 
steps are involved as follows : 

acetyl-P+CoA phosphotransacetylase acetyl-CoA 

> 

' +Pi (1) 

acetyl-CoA+formate Co A- transferase formyl-CoA 



+ acetate (2) 
formyl-CoA -f H2O deacylase formate + Co A (3) 



Sum: acetyl-P-f H2O- 



*acetate+Pi (4) 



Formyl-CoA is synthesized tlirough enzymatic 
transfer of the CoA-moiety from acetyl-CoA to 
formate (reaction 2) . The enzyme catalyzing this 
step is probably identical with the CoA-trans- 
ferase previously shown to catalyze reactions be- 
tween acetyl- CoA and fatty acids with three to 
eight carbon atoms. The deacylase catalyzing 
reaction 3 has little if any activity on other CoA 
derivatives but will catalyze the hydrolysis of 
formylpantetheine. "Wliereas these results point 
to a possible role of formyl-CoA in intermediary 
metabolism, the significance of this substance in 
one carbon metabolism remams to be established. 
In the course of the above investigation it was 
discovered that formyl-thiolesters undergo rapid 
non-enzymatic reactions (at pH 7.0, in dilute 
solutions) with substances containing adjacent 
sulfhydryl, hydroxyl or amino groups or various 



80 



ANlSrUAL REVIEW OF INTRAMURAL RESEARCH 



combinations thereof. Substances with an amino 
group adjacent to a sulfliydryl group undergo 
intermolecular thiolester transfer followed by 
intramolecular S->N migration to form the N- 
formyl derivative. Reaction of formyl-SR with 
bis-amino compounds results in formylation of 
one amino group followed by ring closure with 
the other to produce imidazoledines. Similar 
ring compoimds are formed by interaction of 
formyl-SR with bis-hydroxy amines. These re- 
actions may be helpful in explaining the mecha- 
nism of the enzymatic reactions of formyl 
thiolesters. 

Another outgrowth of the above study was the 
observation that several times re-crystallized prep- 
arations of tetrahydrofolic acid (THF)-for- 
mylase isolated from C. cylindros'porum catalyze 
the formation of formyl hydroxamate when in- 
cubated with ATP, formate, and neutral hydrox- 
ylamine. It is anticipated that further studies of 
this formate activating activity may help to 
elucidate the mechanism of the THF-formylase 
enzyme; however, the possibility that a formate 
activating enzyme is a contaminant in the crystal- 
line THF-formylase preparation has not been 
excluded. 

Carbon Dioxide Activation". Clostridium ther- 
moaceticimi and several other strains of anaerobic 
bacteria have the capacity to synthesize acetate by 
a mechanism in\'olving the condensation and re- 
duction of two molecules of CO2. In an effort to 
determine the nature of the C02-activation that 
is probably involved in this process, the enzymatic 
mechanism is being investigated with cell-free ex- 
tracts of 0. therinoaceticum. Treatment of such 
extracts with Sephadex, charcoal or Dowex-1, re- 
sults in the loss of ability to fix C"02 into acetate. 
This ability is restored by the addition of pyru- 
vate, DPN+, CoA and ATP and as yet other un- 
identified factors in boiled cell extracts of the 
organism. 

Carboxtiation or Acettl-CoA. Malonyl-CoA is 
a critical intermediate in the biosynthesis of long 
chain fatty acids and of certain aromatic com- 
pounds. Malonyl-CoA is formed enzymatically 
by carboxylation of acetyl-CoA in a complicated 
reaction that involves interaction of CO2, acetyl- 
CoA, ATP and a biotin-enzyme complex. It is 
assumed that a biotin-COo-enzyme complex is the 
reactive cai'boxylation intermediate. Extending 



previous studies in this and otlier laboratories 
showing that citrate stunulates fatty acid synthe- 
sis by animal enzyme preparations, it has now 
been established that the citrate effect is concei-ned 
solely with the conversion of acetyl-CoA to 
malonyl-CoA. With purified particulate enzyme 
preparations derived from rat adipose tissue, up 
to 20-fold stimulation of the carboxylation reac- 
tion by citrate is observed. Although other Kreb- 
cycle intermediates produce activation also, citrate 
is by far the most active substance tested. Experi- 
ments with C^Oo show that citrate activation is 
not associated with incorporation of isotopic 
carbon into the citrate molecule. Further experi- 
ments of the mechanism of the citrate effect are in 
progress. 

Two-Garbon Metabolism 

Fatty Acid Synthesis. The first step in the syn- 
thesis of fatty acids involves a condensation of 
malonyl-CoA with acetyl-CoA, accompanied by a 
release of the imesterified carboxyl group of 
malonyl CoA as CO2 and the fomiation of an en- 
zyme bound ^-keto thiolester derivative. Reversi- 
bility of this reaction results in the exchange of 
CO2 with the carboxyl group of malonyl-CoA: 



ESH-FHOOCCHsCOSCoA + CILCOSCoA 
^CHaCOCHsCOSE + COo -F2CoASH 
E 



(5) 



Two protein fractions derived from extracts of 
C. hluyveri are needed to catalyze the overall re- 
action. One of these is stable to boiling for 30 
minutes, and is stable to 0. IN acid or base for 30 
minutes at 25°C. The other fraction contains an 
SH-enzyme that catalyzes a thiol transacylase 
reaction with free CoA or suitable analogues. 
This reaction was measured by the incorporation 
of P^^-CoASH into malonyl CoA according to the 
reaction : 

Malonyl-CoA +P=2-CoASH 

^malonyl-CoA-P^^-fCoASH (6) 

Acetyl-CoA, propionyl-CoA, and butyry]-CoA 
can substitute for malonyl-CoA in this ester in- 
terchange reaction whereas succinyl-CoA, methyl- 
malonyl-CoA, hexanoyl-CoA and octanoyl-CoA 
cannot. It remains to be determined if the ex- 
change reactions observed with tlie lower fatty 
acyl CoA dei-ivatives are catalyzed by tlie same 



NATIONAL HEART INSTITUTE 



81 



enzyme that catalyzes the malonyl-CoA — P^'^ — 
CoASH exchange. Two CoASH analogues, 
pantetheine and phosphopantetheine, can replace 
CoASH in the exchange reactions ; however, other 
mercaptans such as 2-mercaptoethanol and thio- 
glycoUate are not able to do so. 

In confirmation of Lynen's experiments with 
yeast enzyme preparations, it has been established 
that enzyme bound labeled ;8-ketoacid is formed 
when eitlier 2-C"-malonyl-CoA and hesanoyl- 
CoA or l-C"-hexanoyl-CoA and malonyl-CoA 
are incubated with large amounts of exchange 
enzyme preparation from G. hluyveri. 

Evidence that the malonyl-CoA-C02 exchange 
reaction represents an essential step in the syn- 
thesis of long chain fatty acids is obtained from 
the following observations: 1) two protein frac- 
tions that are required for the exchange reaction 
are required also in conjunction with other en- 
zymes for fatty acid synthesis by enzyme prepara- 
tions of C. hluyveri; 2) the exchange enzyme is 
purified coincidentally with the purification of the 
overall fatty acid synthetic activity of enzyme 
preparations derived from mammalian adipose 
tissue; 3) the exchange enzyme is present in all 
preparations thus far examined (three strains of 
bacteria, mammalian adipose tissue, and yeast) 
that catalyze the synthesis of long chain fatty 
acid; 4) reduced triphosphopyridine nucleotide, 
the electron donor in fatty acid synthesis, is a 
strong inhibitor of the exchange reaction. This 
inhibition is presumed to be caused by reduction 
of the /?-keto acid intermediate thereby making it 
unavailable for the reverse of reaction (5). 

In addition to the two protein fractions needed 
to catalyze the exchange reaction, a third protein 
fraction is needed to catalyze the synthesis of 
saturated fatty acids from malonyl-CoA, acetyl- 
CoA and TPNH. Following treatment of the 
purified fractions with charcoal, they must be sup- 
plemented with flavins (FMN or FAD or ribo- 
flavin) and with an unidentified cofactor present 
in boiled cell extracts in order to catalyze fatty 
acid synthesis. 

In contrast to the mechanisms used for the 
synthesis of long chain fatty acids in C. hluyveri, 
it has been shown that the synthesis of butyrate 
by this organism does not involve malonyl-CoA as 
an intermediate. Instead butyrate is formed by 
the condensation of two-moles of acetyl-CoA with 



production of acetoacetyl-CoA which is reduced 
to butyryl-CoA. 

The synthesis of long chain fatty acids from 
malmonyl-CoA and acetyl-CoA by rat adipose 
tissue is catalyzed by a particulate enzyme com- 
plex. During extensive purification of this par- 
ticulate system, the ;8-keto-fatty-a.cyl-CoA re- 
ductase activity (but not enoyl-CoA hydrase or 
a-;8-imsaturated fatty acyl-CoA reductase activ- 
ity) is purified in parallel with the overall fatty 
acid synthetic system. Furthermore, loss of 
/?-keto-fatty-acyl-CoA reductase activity during 
heat and acid denaturation parallels the loss of 
overall fatty acid synthetic activity. These ob- 
servations suggest that the j8-keto-fatty-acyl-CoA 
reductase is an integral part of the biosynthetic 
mechanism. 

When l-C"-acetyl-CoA and malonyl-CoA are 
converted to palmitate in the presence of the 
particulate ezyme and a pool of butyryl-CoA, 
isotope is neither trapped in the butyryl-CoA nor 
does this substance cause dilution of the isotope 
incorporated into palmitate. These results elimi- 
nate from further consideration those theories of 
fatty acid synthesis that postulate an intermediary 
role of free saturated acyl-CoA compounds of 
intermediate chain length. 

Incidental to the studies of fatty acid bio- 
synthesis was the development of a liighly sensi- 
tive method for the separation and identification 
of fatty acid acyl-OoA compounds. This method 
involves conversion of the thiolesters to their 
acetylated hydroxamate derivatives wliich are sub- 
jected to gas-liquid chromatography at elevated 
temperatures. Wlien heated at the top of the 
column, acetylated hydroxamates midergo a Los- 
sen rearrangement to form isocyanates, which 
separate readily upon continued column cliroma- 
tography. 

Ethylene Gltcol Metabolism. Abeles has re- 
ported that the conversion of ethylene glycol to 
acetaldehyde by extracts of Aerobacter aerogenes 
involves an intramolecular rearrangement and a 
loss of water by a mechanism that is obligately 
dependent upon the presence of vitamin B12 co- 
enzyme. This represents the simplest reaction 
thus far observed in which the Bis-coenzyme is 
required and it appears to offer the best oppor- 
tunity to study the mechanism of B12 action at the 



82 



ANNUAL REVIEW OF ESTTRAMURAL RESEARCH 



molecular level. In order to obtain a better ex- 
perimental material for further studies of this 
curious reaction, an anaerobic bacterium was iso- 
lated from the soil that is capable of deriving most 
of its energy from the dissimilation of ethylene 
glycol. The new organism has been characterized 
as a new species belonging to the genus Clostrid- 
ium. In addition to ethylene glycol which serves 
as the major energy source, this organism has nu- 
tritional requirements for 17 different amino acids 
and the three vitamins, biotin, pantethenic acid 
and riboflavin. Of numerous substrate analogues, 
only propylene glycol can replace ethylene glycol 
as an energy source. The fermentation of ethylene 
glycol leads to the formation of equal amounts of 
acetate and ethanol. This is consistent with a 
postulated mechanism in which the ethylene glycol 
is converted to acetaldehyde which undei-goes a 
dismutation to form ethanol and acetate. An 
enzymatic analysis of this system to determine the 
role of vitamin B12 coenzyme is in progress. 

AcETTL Ctanide FORMATION". Cell-free extracts 
of G. hluyveri catalyze the acetylation of amino 
acids in the presence of acetyl phosphate and high 
concentrations of hydrogen cyanide. The curious 
requirements for high concentrations of hydrogen 
cyanide prompted a more intensive study to deter- 
mine its role in the acetylation reaction. It was 
found that an enzyme is present in extracts of 0. 
hluyveri that catalyzes a transacetylation reaction 
between acetyl-OoA and HON to form acetyl- 
cyanide. Acetyl-cyanide is a highly reactive sub- 
stance which, in the absence of an acetyl acceptor, 
undergoes instantaneous hydrolysis; however, in 
the presence of amino acids or mercaptans it 
imdergoes preferential aminolysis or acyl trans- 
fer to form the N-acetylated or tliiolester deriva- 
tives. The HCN-transacetylase was partially 
purified and some of its properties were deter- 
mined. Although the normal substrate for the 
enzyme has not yet been identified, it promises to 
be an interesting new type of acyl acceptor capable 
of forming active acyl derivatives at the energy- 
rich level. Of several substances tested, azide is 
the only compound other than HCN that can 
serve as an acetyl acceptor. 

An outgrowth of the above study was the dis- 
covery that at high concentrations various thi- 
olesters undergo non-enzymatic cyanolysis reac- 



tions with the formation of acetyl cyanide. The 
kinetics of this non-enzymatic reaction have been 
studied in great detail. The data obtained have 
been critical in establishing the role of acetyl 
cyanide as a product of the enzymatic reaction. 

Metabolism of Heterocyclic Compounds 

EiBOFLAviN Degradation. It was previously 
observed that 3,4:,dimethyl-6-carboxy-a-pyrone 
(CsHsOi), urea, and oxamide, appear as products 
during the aerobic dissimilation of riboflavin by a 
pseudomonad isolated from soil enriclunent cul- 
tures. l-Eibityl-2, 3-diketo-l,2,3,4-tetrahydro-6, 
4-dimethylquinoxaline and 3,4-dunethyl-2,3-quin- 
oxalinediol were shown to be intermediates in 
the overall metabolism. Further studies with 
dried-cell suspensions of the bacterium have now 
established the stoichiometry of the main overall 
reaction which is described by the equation : 

C1TH2O6N4+7.5 O2 >6C02+NH3— C— NH= 

o o 

+ NH,— C— C— NH, + CsHsO. + 2H,0 

With prolonged incubation, the a-pyrone 
(C8HSO4) undergoes slow decomposition with the 
uptake of about 4 moles of oxygen, the pi'oduction 
of 4 moles of CO2 and the fixation of one mole of 
endogenous nitrogen which otherwise appears as 
ammonia. In addition to the above organism, 
three new strains of bacteria have been isolated 
thaJt are capable of causing the degradation of 
riboflavin to lumichrome and as yet unidentified 
products. Enricliment cultures capable of cata- 
lyzing the anaerobic dissimilation of riboflavin 
have been obtained also and experiments are in 
progress to isolate the responsible organisms in 
pure culture. 

Nicotinic Acid Fermentation. An organism 
capable of fermenting nicotinic acid with the 
fonnation of stoichiometric amounts of acetate, 
propionate, CO2 and NH3 was formerly isolated in 
this laboratoi-y but it was unfortunately lost. Be- 
cause this fermentation poses a nmnber of ex- 
tremely interesting and fundamental questions, 
another organism capable of fermenting nicotinic 
acid has been isolated from the soil and optimum 
conditions for its gi-owth have been determined. 
Future studies will examine the postulated roles of 
methylmalonyl-CoA, malonyl-CoA, vitamin B,2 



NATIONAL HEART INSTITUTE 



83 



coenzyme and biotin in the dissimilation of nico- 
tinic acid by this organism. The organism will be 
used also in combination with chemical degrada- 
tion procedures to degrade nicotinic acid obtained 
biosynthetically from isotopic precursors. Such 
studies will give an insight into the pathway of 
nicotinic acid biosynthesis. 

Phenazine-I-Caeboxtlic Acid Biosynthesis. 
Studies on the biosynthesis of phenazine-1-car- 
boxylic acid by the bacterium PsevdomoTias aureo- 
faciens have been continued. It was demonstrated 
that all carbon atoms of this pigment are derivable 
from glycerol. The fact that lysine is also re- 
quired, although lysine carbons are not incorpo- 
rated into the pigment, suggests that the ring 
nitrogen atoms are obtained from lysine. The 
possibility that anthranilic acid is a precursor was 
eliminated. A pool of shikimic acid reduces the 
incorporation of glycerol carbon into pigment 
and is therefore tentatively assumed to be an 
intermediate. 

Metabolism of Amino Acids 

Gltcine Eeductions. Insight into the mecha- 
nism of anaerobic phosphorylation associated with 
the reductive deamination of glycine has been 
sought through studies with cell-free extracts of 
Clostridium lento futrescens. With cell-free ex- 
tracts prepared by means of the French pressure 
cell, DPNH serves as the ultimate electron donor 
for glycine reduction in a flavin mononucleotide 
and disulfide-dependent series of reactions. The 
DPNH-linked enzyme system is relatively un- 
stable; aging results in extracts in which only 
1,3-dimercaptopropanol or similar dimercaptans 
can serve as electron donors. The complexity of 
the overall reaction is indicated by the require- 
ments for at least four protein fractions in addi- 
tion to the various coenzymes. As yet no inter- 
mediates have been identified, but it has been 
established that ATP formation and ammonia re- 
lease precede acetate formation. The glycine 
reductase portion of the reaction sequence re- 
quires relatively high ammonium ion concentra- 
tions for activity. 

Ltsine Degradation^. Previous studies have 
shown that lysine degradation by Clostridium 
sticklandii and a newly isolated organism obtained 



from lysine enrichment cultures leads to the for- 
mation of one mole each of butyrate and acetate 
and to two moles of ammonia. It has now been 
established that in fermentations catalyzed by 
cell-free extracts of the new bacterium the acetate 
is derived from carbons 1 and 2 of lysine and that 
butyrate comes from the remainder of the mole- 
cule ; carbon 6 of lysine becomes carbon 4 of buty- 
rate. The overall reaction is sensitive to a num- 
ber of metabolic inhibitors including those that 
inhibit electron transport phosphorylation at the 
flavin level. Treatment of cell-free extract with 
charcoal results in a loss of activity which can be 
partially restored by the addition of boiled cell 
extract or a mixture of DPN, CoA, vitamin Bi2 
coenzyme, pyruvate and 1,3-dimercaptopropanol. 
This interesting enzymatic system promises to be a 
fruitful medium for further studies of electron 
transport phosphorylation and the mechanism of 
vitamin B12 coenzyme action. 

■y-AMiNOBUTTRATE FERMENTATION. The Conver- 
sion of y-aminobutyrate to butyrate during 
fermentations by Clostridium aTnirwbutyricwm 
involves the intermediary formation of y-hydroxy- 
butyrate by a complicated series of reactions (see 
annual report, 1960) . Continuing this study, evi- 
dence has been obtained to support the reaction 
mechanism postulated to explain the further con- 
version of y-hydroxybutyrate to butyrate. Using 
the pantetheine analogues of the normal CoA 
intermediaries, the following enzymatic steps have 
been observed to occur in cell-free extracts : The 
dehydration of y-hydroxybutyryl-pantetheine to 
crotonyl-pantetheine ; the isomerization of vinyl- 
acetyl-pantethteine to crotonyl-pantetheine; the 
DPNH-linked reduction of crotonyl-pantetheine 
to butyryl-pantetheine ; and the conversion of 
acetyl-CoA to acetate by phosphotransacetylase 
and acetokinase activities. Although dinitro- 
phenol inhibits the overall reaction and crotonyl- 
CoA reductase, efforts to demonstrate electron 
transfer phosphorylation with either process have 
not been successful. 

Metabolism of Onium Compounds 

Choline Fermentation. Eesearch has been con- 
tinued on the intermediaiy metabolism of choline 
by a newly isolated anaerobic bacterimn belong- 
ing to the genus Clostridium. Balance studies 



643351—62- 



84 



ANlSrUAL REVIEW OF INTRAMURAL RESEARCH 



with growing cultures show that choline is con- 
verted to trimethylamine, acetate, and ethanol 
according to the overall equation : 

+ + 

2 ( CH3 ) sNCHsCH.OH + H2O - 2 ( CH3 ) 3NH 

+ CH3COOH+ CHsCH.OH 



The stoichiometry of tliis fermentation is there- 
fore similar to that previously observed for the 
choline fermentation of Vibrio cholinicios. How- 
ever, the new organism is a better experimental 
medium to study the enzymatic mechanism since 
all of the enzymes needed for the overall fermen- 
tation are readily obtained in a soluble form and 
are therefore amenable to fractionation. Suspen- 
sions of aged dried cell preparations frequently 
catalyze the fermentation of choline only after 
a long lag period. After the lag period there is 
a substance in the extracellular solution which 
when added to a fresh suspension of tliese cells 
will promote fermentation without a lag period. 
Some properties of the "lag-reducing" activity 
have been determined. Isolation of the substance 
is in progress. Preliminary studies show that the 
fermentation by cell-free extracts is inhibited by 
iodoacetamide, and by compounds known to in- 
hibit electron transport coupled phosphorylation ; 
i.e., 2,4-dinitrophenol, 2,4-dinitrocresol, sodium 
amytal and quinoline-N-oxide, but not antimy- 
cin A. Following aging and treatment with 
charcoal and Dowex-1-acetate, the enzyme prep- 
arations are partially resolved of cofactors and 
can be reactivated by the addition of CoA, ADP, 
Fe'^^ Mg+* or Mn++, and by a heat labile protein 
fraction that is present in the extracellular solu- 
tion of dried cell suspensions which have been 
previously incubated witli choline. This factor 
is not identical with the "lag-reducing" factor 
present in the extracellular solution. 

Fermentation of Sttlfonium Compounds. In 
an attempt to determine if the high energy re- 
leased during hydrolytic cleavage of the sulf onium 
bond can be used for biosynthetic purposes, an 
anaerobic bacterium capable of growing on di- 
methyl-;8-propiothetin (DMPT) as a major 
source of carbon was isolated from the soil. It 
has been established that the fermentation of 
DMPT is described by the overall equation : 



3 (CH3) ,S— CH^CH.COOH-f 2H,0 

- 2CH3CH2COOH + CH3COOH + 3 ( CH3 ) 2S 

+ C0, + 3H* 

Washed cell suspensions also ferment a-alanine, 
/3-alanine, acrylate and lactate to give propio- 
nate and acetate in a ratio of 2:1. Pyruvate is 
fei'mented to produce propionate and acetate in 
a ratio of 1:2. These results therefore suggest 
that this organism is related to C. pi'opionicum 
which carries out similar fermentation of the 3 
carbon compounds. Preliminary enzyme studies 
indicate that DMPT is activated to form the CoA 
derivative which undergoes cleavage to form 
acrylyl-CoA and dimethyl sulfide. The acrylyl- 
CoA is reduced to propionyl-CoA or alternatively 
is aminated to form ;8-alanyl-CoA. It is hoped 
that further studies with this organism will help 
to elucidate the mechanism of the propionate 
formation by anaerobic metabolism. 

Transsulfuration 

Studies on the mechanism of sulfur transfer 
between homocysteine and cysteine have been con- 
tinued. A cystathionine cleavage enzyme iso- 
lated from an me-2 mutant of N eurospora has 
been shown to catalyze the decomposition of cer- 
tain disulfides (L and meso isomers of cystine and 
homocystine) . A similar reaction occurs also in 
animal tissues and may represent the major path- 
way for disposing of tliese compounds within the 
cell and may have relevance to cystinosis and cys- 
tinuria. The evidence obtained indicates that 
disulfide cleavage occurs by a ;8-disulfide elimina- 
tion reaction. Strongest support for this conclu- 
sion is the successful trapping of an unstable 
alkyl hydrogen disulfide intermediate with iodo- 
acetate, and identification of the reaction product 
as the mixed disulfide of cysteine and thioglycollic 
acid. 

With regard to tlie cysteine -^ homocysteine 
process itself, the work has served so far more to 
raise doubts about hitherto postulated pathways, 
rather than to supply an acceptable alternative. 
The reversible process has been thought, for many 
years, to be mediated by enzymes catalyzing ir- 
reversible reactions, 2 cleavages and 2 condensa- 
tions; i.e., cysteine +homoserine (homocysteines- 
serine) — >cystathionine->cysteine+a - keto - butyr- 
ate (homocysteine-l- pyruvate). Only one cleav- 
age enzyme was found in Neurospora, which 



NATIONAL HEART INSTITUTE 



85 



catalyzes a heterogeneous reaction, decomposing 
cystathionine 10% by ;8-elimination and 90% by 
y-elimination. Moreover, only the same mimodi- 
fied enzyme can be found in mutants supposedly 
blocked in cleavage to homocysteine, and in con- 
densation from cysteine. 



LABORATORY OF CHEMISTRY OF NAT- 
URAL PRODUCTS 

The work of the Laboratory may be sum- 
marized in four areas. These are (1) the devel- 
opment of gas phase chromatographic tecliniques 
for the qualitative and quantitative analysis of 
biologically important substances, (2) investiga- 
tion of the isolation, structure, properties and 
biogenesis of plant alkaloids, (3) studies of the 
components of the kallikrein-kallidinogen-kalli- 
din system, and of the chemistry of human 
polypeptide vasodilators, and (4) consultative 
and informal collaboration with various intra- 
mural research groups of the National Institutes 
of Health seeking the specific knowledge and 
equipment of the Laboratory for application to 
their particular problems. 

Gas Phase Chromatographic Methodology 

Research in gas phase chromatography has de- 
veloped along several lines of specific interest. 
With liquid phases developed earlier in the Lab- 
oratory, new classes of compounds were studied 
for gas cliromatographic behavior. These in- 
cluded the vitamins and provitamins D2 and D3, 
sugars (protected as acetyl derivatives), urinary 
l7-ketosteroids, epinephrine and indole metab- 
olites. The sapogenins and steroidal amines, 
tomatidine, solasodine and many of their deriva- 
tives have been separated and identified by this 
method. 

Wliile the extreme sensitivity and high resolv- 
ing power of gas phase chromatography are of 
considerable significance, chromatography of 
larger samples (over 0.5 mgm) has not been par- 
ticularly successful. Progress has been made in 
increasing the amount of material that can be 
chromatographed, maintaining resolution com- 
parable to that of the analytical columns. Sev- 
eral steroid and alkaloid separations have been 



successful using 7-25 mgm samples of 5-com- 
ponent mixtures. 

A third research area in gas phase chromatog- 
raphy has been concerned with the development 
of new liquid phases to provide greater selec- 
tivity in dealing with compomids difficult to 
separate under normal conditions. Neopentyl 
glycol-succinate (NGS) and a fluorinated alkyl 
silicone polymer (QF-1) have proved to be 
promising new liquid phases for this purpose. 
Additional selectivity for the separation of 
closely related compounds has been achieved 
through the use of specific hydroxyl derivatives, 
either trifluoroacetyl esters or trimethylsilyl 
ethers of the alcohol function. The use of two- 
component phases and segmented packings has 
shown promise in exhibiting specificity towards 
various functional groups, making it possible to 
distinguish between steroids differing in type and 
degree of substitution. 



Alkaloid Work 

Minor alkaloids have been isolated from Or- 
mosia jamaioensis and Cassia excelsa. Astro- 
casitie, C20H24N2O, has been isolated from Astro- 
casia pliyllantoides (Euphorbiaceae). Structural 
studies in the major alkaloids of Ormosia, and 
Cassia are continuing. 

In spite of the chemical and pharmacological 
interest in alkaloids for hundreds of years, no 
satisfactory answers have been found to the ques- 
tions of why a given plant makes alkaloids or in 
what manner. Studies are continuing in this Lab- 
oratory to determine the mechanism of alkaloid 
formation in plants. Feeding radioactive tyrosine 
and phenylalanine to various plants of the Ama- 
ryllidaceae has yielded radioactive alkaloids. No 
"scrambling" of the label was observed and it was 
concluded that the C6-C2 fragment of these amino 
acids is incorporated intact into the alkaloid. 
Prior to this discovery, an alternate theory, that 
condensation products of shikimic and prephenic 
acids produced the alkaloids was accepted by some 
investigators. Radioactive compounds, structur- 
ally more complex than the amino acids, have been 
prepared in the laboratory and fed to selected 
plants to study the individual steps by which the 
alkaloids are synthesized in the plant. 



86 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



The Kallikrein-Kallidinogen-Kallidin System 

The vasodilating substance kallikrein owes its 
action to the formation of the physiologically ac- 
tive polypeptide kallidin. The latter compound 
is formed when kallikrein acts on kallidinogen, 
a component of human plasma. Work directed to 
the isolation of kallikrein, kallidin and kallidino- 
gen has been carried on as a joint study with the 
Laboratory of Cardiovascular Physiology. 

The two kallidins, I and II, isolated from hu- 
man plasma after reacting with human urinary 
kallikrien, as described in the 1960 report, have 
been tentatively identified. Kallidin I could not 
be distinguished from the nonapeptide, bradykinin 
(H. Arg. Pro. Pro. Gly. Phe. Ser. Pro. Phe. Arg. 
OH). Kallidin II was identified as the deca- 
peptide H. Lys. Arg. Pro. Pro. Gly. Phe. Ser. Pro. 
Phe. Arg. OH by degradation and by synthesis 
(the latter by E. D. Nicolaides) . Studies are being 
made to determine the physiological role(s) of 
these peptides. 

Highly purified preparations of human urinary 
and pancreatic kallikreins have been obtained. 
Both were homogeneous in the ultracentrifuge. 
The former was also electrophoretically homogene- 
ous, whereas the latter showed two bands. 

Work is in progress to isolate pure human 
plasma kallidinogen, the protein on which the kal- 
likreins act to form the kallidins, in order to deter- 
mine unambiguously the nature of the peptide 
products resulting from the action of pure kalli- 
kreins and to do chemical studies on the kallidino- 
gen molecule before and after enzymatic reaction. 

Miscellaneous Informal Collaborative Research 

In addition to conducting the research cited 
above, a significant amount of time and effort has 
been spent by several members of the Laboratory 
to help other scientists of the National Institutes 
of Health with specific problems. Our help has 
been sought primarily in three areas: (1) Large 
scale processing of microorganisms, plant mate- 
rials, biological fluids, glands, and culture media. 
(2) Assistance in spectroscopic analysis and inter- 
pretation. (3) Advice in the construction and 
repair of gas phase chromatography equipment, 
in the preparation of satisfactory column pack- 
ings and in preliminary studies to determine the 



feasibility of gas phase chromatography for the 
particular problem of the investigator. Consul- 
tative work of this type provides a stimulus for 
members of our Laboratory to bi-oaden their re- 
search outlooks. 



LABORATORY OF CHEMICAL PHARMA- 
COLOGY 



Interaction of Drugs With Physiological and 
Biochemical Function 

The bridge between biochemistry and physiol- 
ogy is the biochemical-physiological interactions 
which explain control mechanisms. Since drugs 
change the quantity, but not the quality of fmic- 
tion, they must act, directly or mdirectly, on these 
control mechanisms. Thus diiigs must pertui'b 
control mechanisms in the same way that these 
are affected by changes in environment. It is even 
useful to regard many diseases, at least sympto- 
matically, as disturbances of regulatory mecha- 
nisms. 

Function is regulated by changing the level of 
hormones at reactive sites. But such regulation 
can be achieved only if the honnone is in a stored 
form. The storage site or neurochemical trans- 
ducer is acted on by the nerve impulse which is 
transmutted into free hormone; this in turn is 
transformed into a change in activity of the next 
nerve cell or of the end organ. Thus the body 
reacts to the stimulus or to a changing environ- 
ment through actions on these neurochemical 
transducers. It follows that one of the basic prob- 
lems in biology is the nature of these units which 
synthesize, store and release substances like nor- 
epinephrine (NE) and serotonin (5HT). 

Amine Storage Site 

Our studies now provide a reasonable picture 
of a unit which synthesizes, stores and releases 
NE or 5HT. We have shown that the amines 
in cells are stored in two pools. One, a mobile 
pool, is separated from monoamine oxidase 
(MAO) and from reactive sites by an active trans- 
port system Avhich maintains the amine inside a 
lipoid membrane. Eeserpine acts by inhibiting 
this transport system. The second, a reserve pool, 
appears to be a complex of amine in granules. 



NATIONAL HEART INSTITUTE 



87 



The nerve impulse causes amine in the mobile 
store to be discharged onto the active site. MAO, 
which surrounds the membrane, has the key role 
of controlling the content of stored amine in the 
face of continuous synthesis. The enzyme facili- 
tates diffusion by inactivating the amine that dif- 
fuses through the membrane; in this way the 
steady-state level is maintained well below satura- 
tion of the transport system. The scheme answers 
such questions as: (1) How reserpine depletes the 
tissue of its amines though it does not act on 
isolated granules. (2) How a pool stored by active 
transport coexists with amine-containing gran- 
ules. (3) How the stored amine is separated 
from reactive sites and IVIAO. (4) Selfregula- 
tion of brain content of amines. (5) Else in brain 
levels after MAO blockade. 

Control of Function hy Drugs Acting on Storage 

Sites 

Viewing the synthesis, storage, physiological re- 
lease, metabolism and the reactive site as a single 
control unit, permits a broad picture of drug 
action. NE and 5HT control units are affected 
by drugs as follows: (1) Drugs may mimic 
amines : Neosynephrine mimics NE at peripheral 
sympathetic sites, amphetamine and LSD at cen- 
tral sites. We are searching for a drug that 
mimics 5HT centrally and has the same relation 
to 5HT that amphetamme has to NE. A new 
benzoquinolizine (P-2647) might be this drug; it 
exerts some central reserpine-like effects but has 
little effect on 5HT content; however, a more 
potent substance would be needed to be of clinical 
value. 

(2) Drugs that block the action of amines: 
Dibenamine blocks NE action peripherally, chlor- 
promazine in brain. With respect to 5HT, des- 
methylimipramine, the antidepressant used in 
endogenous depression, might possibly be a 5HT 
antagonist in brain. 

(3) Drugs that block amine metabolism: A 
number of MAO inhibitors raise brain levels of 
both amines biit the excitation in rabbits is asso- 
ciated with elevation of NE and not 5HT. The 
importance of NE in the action of these inhibitors 
was shown rather definitively by giving MO 911 
(Abbott), a new non-hydrazine inhibitor, after 
reserpine. Reserpine sedation was soon reversed 
but this action was related to NE levels which 
rose later than those of 5HT. 



(4) Drugs that block synthesis: (a) Decar- 
boxylase inhibitors : These block formation of both 
5HT and NE since Dopa and 5 HTP are de- 
carboxylated by the same enzyme. However, 
decarboxylase can be blocked by 90% or more 
without affecting syntliesis of NE. Apparently 
formation of this enzyme must be blocked virtu- 
ally completely before the brain stem dopamine 
level is low enough to affect NE formation, 
(b) Dopamine hydroxylase inliibitors: Since 
a-methyl-m-tyramine is a substrate for the en- 
zyme, we tested the isostere NSD-1034 in hopes 
it might inhibit but not be a substrate. This 
compound appears to be the first agent yet found 
that blocks NE formation in vivo. Doses of 40 
mg/kg block formation of NE in vivo at the dopa- 
mme hydroxylase step without affecting 5HT 
synthesis. Dr. Udenfriend has tested this drug 
for us in vitro and has shown it to be the most 
active inhibitor yet developed. Another isostere, 
NSD-1024, seems even more potent. However, 
NSD-1034 lowers NE level only slightly in one 
hour. Thus blockade of NE synthesis probably 
will not elicit pharmacologic effects rapidly since 
stores must first be utilized. 



HO 

L 



<z 



H 

C— N— NH2 

I 
H CHs 



NSD-1034 



HO 



> 



CHjONHj 



NSD-1024 

(5) Blockade or activation of physiological re- 
lease mechanism : Bretylium is a quaternary sym- 
patholytic agent that lowers blood pressure by 
preventing the release of NE by nerve impulses. 
Our studies indicate that guanethidine, another 
sympatholytic agent, appears to activate the 
process whereby the nerve impulse releases NE 
specifically onto receptor sites; as a result NE is 
depleted peripherally. Pretreatment with bre- 
tylium prevents the depletion of heart NE by 
guanethidine but not reserpine. In addition 
guanethidine causes considerable sympathomi- 
metic activity during the NE release phase, while 
reserpine does not. These results suggest that 
reserpine impairs storage so that amines spill over 
onto MAO, while guanethidine causes release di- 
rectly onto active sites. 



88 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



(6) Blockade of storage processes: (a) Eeser- 
pine and analogues : Eeserpine inhibits the active 
transport processes which maintain NE and 5HT 
in their mobile pools. As a result, the stores are 
depleted. Synthesis remains unabated but release 
is no longer controlled by nerve impulses. The 
physiologic effects of reserpine will depend on the 
resultant amine level at active sites; this in turn 
will depend on the balance between synthesis and 
disappearance. Thus the level of NE at periph- 
eral nerve endings is too low to produce an 
effect and a deficiency results. In contrast, un- 
controlled release of 5HT in the gut results in a 
continuous level at receptors wliich control peri- 
stalsis. Evidence now indicates that reserpine 
does not elicit a deficiency of either amine at their 
active sites in brain because the blood-brain bar- 
rier prevents their rapid diffusion into the blood 
stream, (b) Alpha-methyl-m-tyrosine: This syn- 
thetic amino acid selectively I'eleases catechola- 
mines. It does not act fer se but through its 
decarboxylated product a-methy-m-tyramine and 
the side chain hydroxylated derivative. Like re- 
serpine, MMT is a "hit and run" drug, the action 
lasting long after MMT or its decarboxylated 
products have left the body. Of great potential 
importance in the development of new drugs is 
tlie observation that the sedative Kauwolfia alka- 
loids and the benzoquinolizines are derivatives of 
ot-methyl-m-tyramine. (c) Competitive inhibi- 
tors of storage processes: We have shown that a 
number of drugs including desmethylimipramine 
(DMI) and cocaine, antagonize the uptake of low 
levels of NE by tissues and thereby prolong its 
action. Even though DMI inhibits at a concen- 
tration of 10"^° M it does not release measurable 
amounts of NE and the inference has been made 
that release and uptake of amines are different 
processes. By kinetic studies we have shown that 
these drugs compete with NE for the storage 
process. Since NE level is very high inside 
storage sites, only a small amount is released. 

Eole of 5HT and, NE in Brain 

The question of whether reserpine action is due 
to its effects on the storage of NE or of 5HT must 
be answered before reserpine can be used to dis- 
close the role of brain 5HT. A number of agents 
that selectively block NE storage together with 
a mass of pharmacological and physicochemical 



data has definitely eliminated loss of NE stores 
as an important factor in central effects of 
reserpine. 

If resei-pine action were shown to be due to the 
effects of uncontrolled 5HT release, a great step 
would be taken in elucidating the role of the in- 
dole. Our studies show a quantitative relation- 
sliip between central actions of reserpine, SU 9064 
and Ro 4-1284. In these studies the central action 
is determined by five criteria. Each dose of drug 
is given to many mice and the change in brain 
5HT is related to central activity expressed as 
the number of animals that display all 5 signs. 
The results show a close relationship between the 
degree of central activity and lowering of brain 
5HT even when NE stores are first depleted by a 
selective NE releasing agent. 

The close association of sedation to blockade of 
5HT storage suggests a causal relationship and 
lends support to the view that the action of reser- 
pine does reveal the role of 5HT in the brain. 

Further insight into the role of the brain amines 
was provided by physiologic effects attained by 
their selective release after blockade of INIAO. In 
mice the amines trapped in brain produce I'esults 
which indicate that NE and 5HT integrate dif- 
ferent neuronal systems. Thus, after release of 
NE by MMT, the animals behave as though given 
a super-active amphetamine and dash around, 
perfectly coordinated, at express speed for several 
hours. "VVlien both 5HT and NE are released, far 
less excitation results. Wlien only 5HT is re- 
leased (by first depleting NE with MMT, then 
giving a MAO inliibitor and reserpine) the ani- 
mals show no excitation. From these results it 
appears that effects of 5HT and NE released onto 
their respective reactive sites produce opposite 
responses. Presumably they act on different neu- 
ronal systems. 

Amines as Modulating Agents 

Studies indicate that the NE present in sym- 
pathetic ganglia acts in a local feedback mecha- 
nism to modulate the effects of acetylcholine and 
to prevent the uncontrolled firing of ganglionic 
cells. These results may be a lead in looking for 
the cause of some kinds of hypertension. Further 
studies have shown that the effects of guanethidine 
on ganglionic transmission are erratic because tlie 
release of NE by guanethidine is erratic. NE is 



NATIONAL HEART INSTITUTE 



89 



not present in parasympathetic ganglia, but the 
presence of decarboxylase and MAO suggests that 
some amine is present. 

Our results suggest that a main action or reser- 
pine is on the limbic system which contains largely 
5HT. The results are in accord with the view 
that 6HT in the limbic system is a modulator of 
acetylcholine action The electrophysiological ef- 
fects of reserpine in the limbic system provide 
that first bioelectric phenomenon in brain that is 
associated with a change in brain 5HT. 

Biochemical Behavior 

Central Control of Energy Substrates 

In past years we have studied how the brain 
controls biochemical processes through the hy- 
pothalamic-pituitary-endocrine and the autonomic 
systems. This past year, we have been concerned 
with how these systems interact, especially in 
mobilizing energy substrates. 

(1) Eole of brain and sympathetic nervous sys- 
tem in mobilizing energy substrates : The control 
by the nervous system of the release of free fatty 
acids (FFA) from adipose tissue was shown by 
stimulation of sympathetic fibres innervating 
omental adipose tissue in situ. Electrical stimula- 
tion increases (3-fold) the amount of FFA in 
efiluent blood. The breakdown of triglycerides to 
FFA is exquisitely sensitive to free NE. After 
reserpine, free NE but not nerve stimulation mobi- 
lizes FFA ; after dibenamine, neither nerve stim- 
ulation nor free NE mobilizes FFA. 

In the absence of NE it appears that FFA and 
other energy substrates are not mobilized in phys- 
iological situations-requiring increased energy. 
For example, the effect of depot ACTH in mobi- 
lizing FFA from adipose tissue (and the resultant 
triglyceride deposition in liver) does not occur in 
animals depleted of NE stores ; infusion of NE 2 
hours before ACTH restores the NE stores in 
adipose tissue and the FFA are now released by 
ACTH. Similarly fat pads of animals which 
have been depleted of NE do not respond to 
ACTH or glucagon without addition of NE to 
the incubation mixture. 

(2) Requirements for both corticoids and sym- 
pathetic nervous system in mobilization of FFA : 
Ethanol, morphine and many other dinigs produce 
manifestations typical of pituitary- adrenal stim- 



ulation including a fall in adrenal ascrobic acid, 
a rise in plasma corticosterone, a rise in FFA, and 
deposition of liver triglycerides (TGL). These 
effects do not occur after hypophysectomy or 
adrenalectomy but still occur after adrenal de- 
meduUation. Similar results are produced by 
depot ACTH. If the rats are pretreated with 
dibenamine or Ecolid and then given ethanol, 
morphine or ACTH, the rise in corticosterone 
level still occurs but the mobilization of FFA and 
rise in liver TGL are prevented. This suggests 
that the release of FFA is dependent on both 
an intact sympathetic system and on the presence 
of corticoids. 

The essential role of glucocorticoids in lipid 
metabolism was shown by the following experi- 
ments : a) NE infusion failed to raise FFA levels 
in adrenalectomized rats miless the animals were 
pretreated with cortisone; b) NE added to the 
fat pads of adrenalectomized rats does not mobi- 
lize FFA unless the animals have been pretreated 
with cortisone. Preliminary work indicates that 
the corticoids might be involved in the passage 
of FFA from adipose tissue, while NE is con- 
cerned in breakdown of TGL. 

Central Control of Metabolism 

Processes of conservation and restoration are set 
in motion by reserpine-like drugs. In doses that 
do not deplete adrenal catecholamines in rats, 
these drugs produce a sleep-like state with an in- 
creased parasympathetic output and the result- 
ant economy of fimction. The evidence suggests 
that they activate processes that are superimposed 
on normal function and which might be similar to 
those elicited in sleep and hibernation. 

A dramatic example of the action of reserpine 
in activating conservation processes is provided 
by its effects on energy production. Thus reser- 
pine was found to produce a profound drop in the 
metabolic rate in nonnal and thyroidectomized 
animals. In thyrotoxic animals it rapidly re- 
serves the high metabolic rate to below the normal 
level. This action is not exerted upon the thyroid 
gland but upon the tissues where it counteracts 
the effects of thyroxine. The action of reserpine 
is prevented by pretreatment of desmethylimipra- 
mine, a specific antagonist of the central actions 
of reserpine, showing that the reserpine effect on 
BMR is a central one. 



90 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Destnethylimipramine, a New Antidepressant 

In studies of comjDounds that block the reser- 
pine-induced syndrome, imipramine was found to 
counteract the centx'al actions of reserpine and 
reserpine-like compounds in rats. Its delayed 
onset of action was explained by the slow accumu- 
lation of a metabolite, the monomethylanalogiie 
(DMI) . Unlike imipramine, DMI has no sedative 
action per se; it neither inhibits MAO nor affects 
the release of amines by reserpine. If given be- 
fore reserpine (or other reserpine-like com- 
pounds) it prevents the complete reserpine syn- 
drome including sedation, decreased reactivity to 
stimuli, increased parasympathetic output, de- 
creased basal metabolism, etc. In higher dosage 
it reverses the reserpine syndrome, that is, it pro- 
duces a hyperactivity which is not an increase 
in normal activity but appears to be the op- 
posite of reserpine sedation, that is endogenous 
excitation. 

The rats are not influenced by outside stimuli, 
but act as though controlled by an inner drive. 
They jump from great heights, relentlessly travel 
a treadmill till they drop and ignore the presence 
of other animals, as well as of food and water. 
These effects are elicited by reserpine up to 30 
hours after a single dose of DMI, but if reserpine 
is given first and then DMI, the effects of the 
reserpine are no longer influenced. 

Some experiments are in accord with the view 
that DMI competes with 5IIT for reactive sites. 
Thus: 1) Eabbits given an MAO inhibitor fol- 
lowed by DMI die in hpyerpyrexia. 2) If rats 
are depleted of NE (with MMT) and then given 
DMI, reserpine action is still blocked. 3) DMI, 
in contrast to amphetamine, blocks the jDarasym- 
pathetic actions of reserpine. However, other ex- 
pei'iments indicate that DMI might activate the 
action of NE in brain. Neither concept gives a 
satisfactory explanation for the fact that DMI 
has no antidepressant effect in normal animals. 

The following suggest that the effects of DMI 
may be a link between biochemistry, physiology 
and a brain disease. 1) DMI does not produce 
excitation in normal subjects, but only in those 
having a primary depression. 2) Eeserpine elicits 
an "endogenous" depression which is converted 
by DMI to an "endogenous" excitation. 3) Over- 
dosage in subjects with endogenous depression can 



elicit hypomania. These results suggest that sim- 
ilar central pathways might be involved in endog- 
enous and reserpine-induced depression. 

Passage of Substances Across Membranes 

Ca in Membrane Permeability 

Ethylenediamine tetracetic acid (EDTA) in- 
creases intestinal absorption of lipid-insoluble 
compoimds such as mannitol, inulin, decametho- 
nium, and sulfanilic acid. The non-specificity of 
the effect was indicated by the increased passage 
of inulin-C" from plasma to gut. These results 
suggest that binding of Ca changes the character 
of the boundary. 

Penetration of Drugs Into Cells 

Organic anions enter red cells while cations do 
not. Our studies show no indication of competi- 
tive inhibition, no saturation phenomena, and no 
effects by metabolic poisons, and suggest that 
anions pass into the red cells by a process of simple 
diffusion through positively charged pores. 

Biliary Excretion of Drugs 

The liver transports some quatemaiy am- 
moniiun compounds, e.g., procaine amide ethobro- 
mide, Darstine and benzomethazine, into bile. 
Strangely enough decamethonium and tetra- 
ethylammonium are not concentrated in bile. 

Memhranes Within the CNS 

There is no barrier to substances leaving the 
cerebrospinal fluid (CSF). Various sugars such 
mannitol, sucrose, inulin and dextran (M. W. 180 
to 40,000) injected into lateral ventricle or cis- 
terna magna leave CSF into plasma at same rate, 
presumably via the arachnoid villi. The rate of 
passage appears to be a measure of bulk flow (or 
turnover) of CSF, since the exit of inulin is re- 
lated to CSF hydrostatic pressure. In contrast, 
phenol red and PAH leave the CSF by a special- 
ized transport system. Decamethonium and 
hexamethonium also appear to leave by a special 
mechanism, but the process differs from tlie one 
for acids. 

Preliminary results suggest that highly jiolar 
substances can be trapped inside the blood-brain 
barrier. Thus the amines formed by decarboxyl- 
ation of MMT remain in In-ain long after tliey 



NATIONAL HEART INSTITUTE 



91 



can be found in other tissues. In addition the 
guanethidine that can be forced into brain after 
intracistemal injection remains there for a con- 
siderable period of time. Tliis trapping effect of 
the blood-brain barrier indicates that bulk flow 
between brain and subaraclinoid space is slow. 
This finding can possibly be exploited in therapy. 

Trans'port of Pwrines and Pyrimidines 

Uracil is actively transported in vitro. Strong 
inhibitors have substituents at the 5 or 6 positions 
of the pyrimidine ring, while compounds with 
substituents at 1, 2, 3 or 4 positions were weak 
inliibitors. Hypoxanthine and compounds simi- 
lar to hypoxanthine are the best inhibitors of 
uracil transport, although these compounds them- 
selves are not transported. 

Drug Metabolism 

Antimetabolites Metabolized by Enzymes of 
Intermediary Metabolism 
6-Methylaminopurine (methylated adenine or 
MAP) is one of several methylated derivatives of 
adenine and guanine fomid in trace amounts in 
soluble-ENA. An enzyme in rat liver converts 
adenine to MAP. Since inclusion of N-methyl- 
ated purine m nucleic acid might upset genetic 
coding, studies on utilization were carried out. 
However, the results show that MAP is efficiently 
incorporated into lUSTA, mainly as adenine and 
guanine and only to a slight extent as MAP. In 
fact, in mammalian tissue and tumors it is a more 
efficient precursor of EN^A than adenine. How- 
ever, the first step in the metabolism of MAP does 
not appear to be demethylation. These results 
are in accord with the ^aew that tissues have a 
small pool of MAP and that methylation may 
have an important role in the synthesis of nucleic 
acid, perhaps to transfer adenine from one intra- 
cellular pool to the other. 

Substances Acted on by Non-specifiG Enzymes 
Not Involved in Intermediary Metabolism, 

a) Oxidation and reduction enzymes in micro- 
somes: These important enzymes are involved in 
the metabolism of most drugs. The oxidative 
enzymes utilize O2 from air and act thx'ough a re- 
action of TPNH oxidase, Oo and TPNH to form 
an active hydroxyl donor. Data now indicate that 
the TPNH oxidase is a common denominator in 

643351 — 62 8 



both the oxidation and reduction of drugs. A 
common step to both kinds of reaction is: oxi- 
dase + TPNH > reduced oxidase -1- TPN, then 
in air : reduced oxidase + O2 > "active hydroxyl 
donor". In the presence of an excess of FAD or 
anaerobically : 

reduced oxidase -1- FAD = oxidase 

+ FAD H2 
FAD H2 + nitro compound > amino 
compound + FAD 

TPNH oxidase has been solubilized. In addi- 
tion nitro reductase has been solubilized. These 
might be important achievements, since their 
solubilization has been a barrier to elucidation of 
the mechanism of action. 

b) Mechanisms of inliibition : Since drag com- 
binations are often prescribed, it is important to 
know how one drug affects the metabolism of 
another. The kinetic analysis of microsome oxi- 
dation in vitro has suggested a number of mecha- 
nisms of drug inhibition — ^thus 1) Drug A (mono- 
methyl-d-aminoantipyrine) competitively inliibits 
o-demethylation of drug B (p-nitroanisole) by 
combining with tlie enzyme. However, drags A 
and B are metabolized by entirely different 
enzymes. 

Substances like SKF 525-A, a potent and gen- 
eral mhibitor of drag enzymes, act non-competi- 
tively by unknown mechanisms. 

c) Induced enzyme formation : There are three 
mechanisms by which drags cause an increase in 
drug metabolism. 1) Our work shows that the 
effect of testosterone in increasmg rates of drug 
metabolism in female rats is actually due to the 
anabolic effects of testosterone and can be pro- 
duced by analogues having high anabolic activity 
and low sex activity. These substances do not 
increase synthesis of ascorbic acid and the induc- 
ing effects are additive to the general inducing 
action of drugs. 2) Hydrocarbons like 3, 4-benz- 
pyrene and 3-methylcholanthrene have been shown 
to act through different mechanisms than pheno- 
barbital and pi'esiunably other drugs. 3) The 
general non-specific action whereby many drugs 
induce an increased activity of their own and other 
drag metabolizing enzymes is still obscure. The 
action is not mediated through any known endo- 
crine gland. It is of considerable interest that 
the repeated administration of MER 29 stimulates 
metabolism of acetanilide, hexobarbital, etc., and 



92 



ANlSrUAL REVIEW OF INTRAMUKALi RESEARCH 



presumably its own metabolism. The clinical sig- 
nificance of this finding is miknown. 

Metabolism of Imipramine {Tofranil) 

The lack of correlation between effects and levels 
of imipramine, the antidepressant drug, made us 
suspect an active metabolite. Repeated doses in 
rats results in accmnulation of a compound, identi- 
fied by gas chromatography as the monomethyl 
analogue. 



Deposition of Calcium 

Studies of factors regulating fixation of Ca 
in the aorta and the relationship to cholesterol 
accumulation show: a) On incubation of aortas 
with serum containing Ca*^, there is no uptake 
for 24: hours and 10% of label is taken up in the 
next 48 hours. There is a parallel deposition of 
Ca and P measured chemically. The process is 
temperature dependent and is prevented by pre- 
heating at 80° C. for 10 minutes. Under nitrogen, 
Ca uptake is drastically reduced. X-ray crystal- 
lography and electron microscopy show typical 
hydroxyapatite formations which by histochem- 
ical methods was shown to involve an elastin com- 
ponent, b) Pre-incubation of aortas with various 
enzymes indicates that in contradiction to the 
postulated role of collagen, only elastin was im- 
portant in calcification. 

Plasma contains an inhibitor of calcification, 
probably a protein. 



Deposition of Triglycerides (TGL) 

TGL deposition in liver after the administra- 
tion of ethanol is blocked by hypophysectomy. 
This is reflected by increased incorporation of 
C^* palmitate into TGL at expense of phospho- 
lipids. Wlien ecu is given, TGL deposition is 
only partly blocked by hypophysectomy. A main 
effect of CCI4 is to impair hepatic release of TGL 
from liver to plasma. This is shown by fall in 
plasma TGL chemically determined, and by spe- 
cific activities of TGL fractions in liver and 
plasma after administration of palmitate C". 



Development of New Drugs 

Reserpine Analogues 

Eeserpine is difficult and sometimes dangerous 
to use in prolonged therapy since it exerts a cumu- 
lative action owing to its high affinity for the 
transport system of amine storage sites. The re- 
versibly acting SU 9064 (methyl ether of methyl 
reserpate) was developed in collaboration with 
Ciba Laboratories on the basis of physicochemical 
prognostications. It is now in clinical trial in 
the treatment of psychoses. 

Antidepressants 

We have shown that the action of imipramine 
is mediated through its metabolite, desmethylim- 
ipramine (DMI) . Clinically this drug is effective 
and acts rapidly in primary depression. In col- 
laboration with Geigy, Switzerland, we are look- 
ing for the structural characteristics of active com- 
pounds. We have foimd a number of monomethyl 
analogues of imipramine and chlorpromazine 
which reverse the characteristic reserpine-like 
syndrome. Trials in man will show whether DMI 
is the progenitor of more active antidepressants. 

Dopamine. Hydroxylase Blockers 

Collaborative studies with Smith-Nephew 
(England) have yielded two isosteric analogues 
of NE (NSD-1034, NSD-1024) which block syn- 
thesis of NE at the last step. These drugs are 
potentially of clinical use in producing a selective 
deficiency of NE in certain parts of the body. 

Bemoquinolizines and Other Compounds That 
Might Mimic BET Action 

Structural relationships between reserpine, «- 
methyl-m-tyramine and other amine releasers have 
led us to believe that suitable modification might 
yield a compound which mimics the action of 
5HT in brain. In studies of Pfizer's benzo- 
quinolizines it appeal's that P-2647 might act 
like a weak reserpine, but releases only a small 
fraction of the brain amines. Attempts will be 
made to turn vip more active derivatives. Ana- 
logues and isosteres of a-methyl-m-tyrosine that 
might act in a similar way are being studied in 
collaboration with Hoffmami La Roclie. 



NATIONAL HEART INSTITUTE 



93 



I 



Bretylmm and Guanethidine-Like Drugs. 

In collaboration with Burroughs-Wellconie, we 
are investigating structural requirements for acti- 
vation or blockade of the system that releases NE 
at reactive sites. Small changes in structure con- 
vert one kind of compound to the other, but both 
are potentially useful in hypertension. 

Development of New Methods of Analysis 

The following procedures have been developed : 
1) A simple sensitive extraction procedure to assay 
dopamine in biological material which provides 
a routine procedure since it eliminates the need 
for tedious column chromatography. 2) A colori- 
metric method for the determination of guanethi- 
dine in biological material. 3) Methods to 
determine a-methly-m-tyramine and ct-methyl-/8 
hydroxy-m-tyramine in tissue preparations. 4) A 
sensitive fluorometric method for agmatine, a good 
substrate for diamine oxidase. 5) A modification 
of the procedure of Shore and Olin to assay 
norepineprine in adipose tissue. Preliminary 
studies with paper chromatograms containing imi- 
pramine and metabolites indicate that at the 
temperature of liquid nitrogen these compounds 
have a pronounced phosphorescence. This prop- 
erty is the possible basis for fluorescence assay at 
low temperature. In drug action studies, classi- 
cal methods of enzyme inhibition are often inap- 
propriate since an enzyme may appear to be 
completely blocked in vitro, yet be functionally 
active in vivo. For this reason it has been neces- 
sary to develop methods of determining whetlier 
enzymes are blocked in vivo by measurement of 
rate at which it performs its function in the intact 
organism. Methods have been developed to assay 
monoamine oxidase, dopa-5HTP decarboxylase, 
and dopamine hydroxylase in vivo. 



LABORATORY OF TECHNICAL DEVELOP- 
MENT 

Gas Chromatography 

The work of this year consisted of efforts to 
develop methods for studying biochemical and 
physiological research problems. Primary em- 
phasis continued to be on develojjment of methods 
of analysis by gas chromatography including 



ultrasensitive methods of detection, methods for 
measurement of radioactivity in gas chromato- 
graphic effluents, and methods for applying these 
tecliniques toward the solution of specific bio- 
chemical and physiological research problems in 
which this laboratory collaborated or which this 
laboratory instituted or both. 

The further development of the direct current 
electrical discharge detector for gas-liquid chro- 
matography was continued. It had been shown 
elsewhere that the electrical conductivity of argon 
gas in the presence of a radioactive source and a 
high voltage was highly sensitive to the addition 
of small quantities of organic vapor. Because 
the presence of radioactivity is objectionable in 
many applications, a study was instituted to de- 
termine the conditions necessary to operate this 
device without radioactivity. By varying the 
voltage across the detector chamber, it was 
demonstrated that at low voltages the currents 
obtained are determined by tlie quantity of radio- 
activity present, while at higher voltages this de- 
pendence is lost. At higher voltages the fact that 
the current is independent of the presence of 
radioactivity makes operation of the device with- 
out radioactivity feasible. A detector was de- 
veloped and its use was explored. 

This device was found to operate only at very 
low concentrations of organic vapor. In its effec- 
tive range tliis method is probably the most sensi- 
tive method for detection of organic vapors by 
ionization in argon. However, it is somewhat 
more sensitive than some of the other methods to 
atmospheric gas contaminants and to poisoning 
of electrode surfaces by adhesive anti-corona 
agents such as silicones. 

The stable continuous discharge produced by a 
high direct current (D.C.) voltage in argon and 
helium can be used to replace a radioactive source 
of electrons. The plasma thus produced can be 
used as a conductance sensitive to organic vapors 
and measured with a second set of electrodes. As 
a result, the concentration over which the detector 
can be used is increased to higher levels, and the 
sensitivity to contamination is decreased. This 
detector is now in use in our laboratory. 

The possibility of applying the continuous D.C. 
discharge to the detection of atmospheric gases 
analyzed by gas chromatography was explored. 
The device was found to be quite sensitive to oxy- 
gen, nitrogen, and carbon dioxide. The resDonse 



94 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



and the sensitivity to each of these gases was found 
to be dependent on the carrier gas used, its purity 
in the detector cell, and the nature as well as the 
concentration of contaminants present. 

Wlien argon is used as carrier gas, oxygen re- 
duces its electrical conductivity. Detection of 
parts per million of oxygen is possible. Nitrogen 
in low concentration enhances the electrical con- 
ductivity. Higher concentration reduces it. The 
sensitivity for oxygen is approximately 10-20 
times that for nitrogen. Addition of low concen- 
trations of organic vapor increases the sensitivity 
to each of the atmospheric gases. 

Wlien helium is used as carrier gas, oxygen, ni- 
trogen and carbon dioxide are measurable in the 
parts per billion range. They enhance the con- 
ductivity of pure helium. Small concentrations 
of each of these, however, overload the detector. 
The concentrations at which overloading occurs 
and the maximal conductivity obtainable varies 
with the different gases. It had been frequently 
reported that extensive purification of helium was 
necessary to obtain this effect. In the work de- 
scribed here commercial helium was used to obtain 
this highly sensitive detection of atmospheric 
gases. 

Determination of Blood Gases 

Experimentation with the D.C discharge 
pointed the way to a series of methods for detect- 
ing atmospheric gases. Each of these methods 
is either more sensitive or easier to operate than 
the methods presently in use in analyses of blood 
gases. This higher sensitivity, for example, makes 
it possible to perform analyses on the oxygen con- 
tained in a microliter of blood. 

The problem that has hindered the development 
of gas chromatography for blood gas analysis is 
that of removing the gases from the blood and 
delivering them to the flowing gas stream within a 
narrow time interval. It was fomid possible to 
remove the oxygen from oxyhemoglobin by heat. 
This permitted injection of blood into the inlet 
of a gas chromatography column system directly. 
The results obtained agreed quite closely with 
those obtained from blood oxygen using the con- 
ventional Van Slyke procedure. 

The present effort is directed toward eliminat- 
ing several experimental difficulties, such as those 
caused by coagulation of the blood in the introduc- 



tion system. It is hoped to establish this as a work- 
ing system of analysis and to extend the technique 
to a series of other determinations of physiological 
interest. 

Radioa.ssay : Scintillation Counting 

A method has been developed whereby labelled 
compounds in the effluent of a gas stream are con- 
densed quantitatively in the presence of a scintil- 
lator. The radioactivity is then assayed by 
scintillation counting. 

Two approaches, using this method, have been 
explored. In one, the entire column effluent is 
collected in a single collection device wliich is 
monitored by scintillation counting throughout 
the procedure. This has been used with moderate 
success in studies of fatty acid synthesis. A 
major effort this year has involved extending this 
technique to the assay of sterols for a study of 
sterol synthesis. Unfortunately the sterols and 
the fatty acid esters differ sufficiently in volatility 
to require, at the moment at least, two different 
methods for condensing them. For fatty acid 
esters, the gaseous effluent is passed through a 
cigarette shaped glass cartridge filled with coated 
anthracene crystals. Upon emerging from the 
heated column outlet tube these are sufficiently 
volatile to pass promptly tlirougli the first layer 
of anthracene ciystals. This layer is heated by the 
hot gas and the anthracene scintillates at less than 
optimal efficiency. By addition of a coolant air 
stream to the photomultiplier compartment, the 
successive layers of anthracene where the materials 
are condensed and retained are cooled sufficiently 
to scintillate well. 

Sterols, however, condensed in the heated layer 
of anthracene when this system was used. An 
alternative system was devised whereby the col- 
umn effluent was discharged into the cavity of a 
fumiel containing anthracene ciystals M-hicli were 
kept under suction. The top layer of anthracene 
was cooled by the air passing through it while tlie 
sterols were depositing on it. This sj'stem was 
developed and applied effectively in a study of 
cholesterol biosynthesis. Fatty acid esters, how- 
ever, are too volatile to be retained in the funnel 
in the face of the torrent of gas necessary to bring 
the column effluent into the funnel. A successful 
method for retaining the shorter chain esters in 
this system has not yet been developed. 



NATIONAL HEART INSTITUTE 



95 



The second approach to radioassay by scmtiUa- 
tioii counting has involved collecting the effluent 
of the column in a series of anthracene-filled car- 
tridges for subsequent scintillation counting. A 
careful study of the scmtillation properties of 
anthracene, and of the mechanics of scintillation 
counting using a commercial liquid scintillation 
spectrometer, yielded a method for reproducible 
and quantitative recovery of labelled compounds. 
An automatic fraction collector for this purpose 
was developed. 

The feasibility of collecting equal fractional 
aliquots for radioassay was demonstrated. This 
yielded a high sensitivity radioassay which, of the 
possible approaches, was least likely to allow op- 
erator bias to contribute to error in interpretation 
of the results. 

Radioassay : Ionization Chamber 

This project, initiated last year, first involved 
attempts at enhancing the response of ionization 
chambers for radioassay. It was predictable that 
the current yield per radioactive disintegration 
could be increased by using argon with a trace of 
organic vapor as ionization chamber gas. The 
combination of argon, high voltage, and a source 
of radioactivity has been used as a sensitive de- 
tector for organic vapors. It was shown feasible 
to substitute the radioactivity for the concentra- 
tion of organic vapor as the variable. The sys- 
tem, however, remained sensitive to small changes 
m organic vapor concentration. This made the 
approach less attractive as a method for increasing 
sensitivity, since, in gas chromatographic applica- 
tions, the organic vapor concentration could be 
expected to vary. A second approach was sug- 
gested by the work of Cacace, who used a large 
ionization chamber and thus achieved greater 
yield in ion current from each disintegration, 
while keeping the time constant of the system 
small by purging the chamber with an accessory 
gas flow. This approach was tried with a 
modification. 

To avoid difficulties caused by differing vola- 
tilities of samples and by the operation of insula- 
tors at high temperature, it was decided to 
combust the sample to carbon dioxide and operate 
the ion chamber at room temperature. 

A study of the behavior of this system was 
undertaken to determine its ultimate sensitivity. 



both for routine use in our laboratory and for 
comparison of this approach with that of the 
anthracene scintillation system. Conditions for 
complete combustion of organic vapors in the col- 
umn effluent were determined. Changing the car- 
rier gas used was found to have an effect on the 
result obtained, but the effect was not sufficiently 
large to prohibit the use of any of a number of 
gases should the need arise. Although the work 
is incomplete, it appears that the ultimate limit of 
sensitivity of this system will be determuied at 
least partly by its sensitivity to change in the com- 
position of the gas in the chamber, as well as by 
leakage currents and other more conventional diffi- 
culties with ion chamber measurement. The sensi- 
tivity, however, seems quite high. This work is 
currently being completed. 

Analysis of Free Fatty Acids : Use of Carbon Di- 
oxide as Carrier Gas 

Several possible methods of analyzing free, long 
chain fatty acids were explored. These materials 
emerge from the gas chromatograph as asym- 
metrical peaks, making quantification difficult. 
The degree of asymmetry was fomid to depend on 
the concentration of fatty acids present m the 
colunui, higher concentrations yielding more asym- 
metrical peaks. Although the asymmetry is usu- 
ally attributed to adsorptive binding on active 
sites of the solid support, it was also found when 
the inert material Teflon was used as solid sup- 
port. Addition of phosphoric acid to the liquid 
phase was tried and fomid effective in reducing 
the asymmetry as had been reported elsewhere. 
The latter, however, remained when very small 
quantities of acids were analyzed. Carbon dioxide 
as cai'rier gas was also found to reduce the asym- 
metry of peaks, and, as did the addition of phos- 
phoric acid, to reduce the retentivity of the liquid 
phase of the colmmi. 

These effects of carbon dioxide were not limited 
to the analysis of free fatty acids. Chromatog- 
raphy of sterols and fatty acid esters was simi- 
larly affected. No sacrifice of sensitivity is 
involved in the use of carbon dioxide since the 
hydrogen flame ionization detector is insensitive 
to it. 

The great sensitivity of standard ionization 
chambers to radioactivity was exploited to extend 
the range of analysis of gas chromatographic 



96 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



column effluents for C". A pyrolyzer was placed 
at the end of a fatty acid column and the C"02- 
labeled products of combustion were rmi into the 
ionization chamber at room temperature. The 
ionization chamber was purged with gas at about 
3 times its volume each minute. This simple sys- 
tem was found to effect the conversion of fatty 
acids completely to CO2. It was found that sam- 
ples with activity as low as 150 counts per minute 
could be detected. 

The chromatographic apparatus and methods 
developed have been applied to several metabolic 
studies in collaboration with the Laboratory of 
Cellular Physiology and Metabolism. 

Porphyrin 

Porphyrin analysis by gas chromatography was 
considered for the study of the deposition of en- 
dogenous porphyrins in tissue and tumors. Sev- 
eral porphyrins were chromatographed at tem- 
peratures in excess of 400° centigrade but rapid 
destruction of those porphyrins containing oxygen 
and partial degradation of others indicated that 
the method had severe limitations. Methods of 
extracting porphyrins from tissue and determin- 
ing their fluorescence were explored and developed 
to facilitate a study of the localization of endog- 
enous porphyrins in rats bearing experimental 
Walker 256 carcinosarcomas. If localization in 
the tumor were sufficiently specific, it might pro- 
vide a method for labeling or treating some new 
growths. In the course of studies it was noted 
that tetraphenylporphiaesulfonate was concen- 
trated to a greater extent than hematoporphyrin. 

Blood Flow Measurement 

Ultrasonic, nuclear magnetic resonance, and in- 
dicator dilution methods are being developed to 
facilitate hemodynamic measurements. These 
methods hold some promise of greater stability and 
sensitivity, especially for measurement of lower 
flow rates. 

The ultrasonic blood flowmeter systems cur- 
rently available cannot discriminate low flow rates 
from noise generated by the switching circuits that 
compare upstream and downstream sonic velocity 
to determine flow velocity. A refinement of this 
technique has been developed in which the up- 



stream and downstream velocities are measured 
simultaneously without ambiguity by using a dif- 
ferent carrier frequency for each channel. The 
signals are separated by means of filters. The 
difference in velocities gives a continuous indica- 
tion of flow velocity. This procedure is free of 
errors introduced by comparing signals separated 
in time and obscured by switching noise. 

The system, which has been constructed and 
tested, performs according to theoretical predic- 
tions. Construction of a transducer head suitable 
for application to blood vessels will allow testing 
in vivo. 

The nuclear magnetic resonance flowmeter pro- 
gram is continuing with development of high 
stability, low noise electronic circuits and sensing 
heads. At the present time the system can be used 
to measure flows of the order of 5 to 300 cc/minute 
in a tube of 3 mm inside diameter. Flow in larger 
tubes can be measured with greater ease than small 
tubes. Several of the circuits developed in the 
design of these flowmeters have had novel features. 

Work has been completed on a project for im- 
proving the use of ascorbic acid for the detection 
of cardiac shunts. A platinum electrode on the 
tip of an intracardiac catheter is used to measure 
the redox current which is related to the ascorbic 
acid concentration. The technique has been found 
valuable in detecting shunts during cardiac cathe- 
terization. 



Ultrasonics 

The chemical and physical interactions of ultra- 
sound in various media have been studied to deter- 
mine the extent to which chemical reactions can 
be modified or accelerated by this form of energy. 
The extent to which changes in velocity and at- 
tenuation can be used to measure the progress 
of chemical and enzymatic reactions has also been 
investigated. 

Ultrasound at intensity levels sufficient to cause 
cavitation produces sonichemical oxidations by 
the action of hydroxyl free radicals. Tiypto- 
phan, for example, when irradiated produced for- 
mylkynurenine, Ivynurenine, and 3-hydroxy kynu- 
renine. Irradiation of pthalic and benzoic acids 
produced fluorescent hydrox}flated products. 
This system has been utilized in the development 
of a method for measuring these aromatic acids 



NATIONAL HEAET INSTITUTE 



97 



by the fluorescence of the hydroxy-acids produced. 
The attenuation and velocity of ultrasound was 
measured in a reaction cell to determine the time 
course of enzymatic reactions. It was possible to 
obtain a satisfactory record of the action of inver- 
tase on sucrose. Starch and alpha amylase have 
been shown only to give a satisfactory signal. 
The system is considered to have reasonable poten- 
tialities for the study of reaction where no good 
spectrophotometric metliods are available. 

Theoretical Analysis of Transport 

The earlier work on integral equation methods 
in biological transport problems has been con- 
tinued and expanded. Some of the fundamental 
notions in biological transport theory have been 
more closely examined. This has resulted in a 
compact set — theoretic definition of input-output 
systems. In this formulation a biological event 
is taken to have an undefined but intuitively under- 
stood meaning. Everything else is defined in 
terms of event and the primitives of set theory. 
This leads to the definition of a biological system 
as a set of ordered "stimulus-response" or "input- 
output" pairs in which each member of the pair is 
a set of biological events. This definition has the 
advantage that mathematical structure can be 
introduced into the analysis in a systematic way. 
For example, a linear biological system is repre- 
sented as a linear operator defined on a linear 
vector space whose members correspond to states 
of the system. Such an operator representation 
provides a general and compact symbolic language 
divorced from computational details. For ex- 
ample, for states distributed discretely in time 
the operators, symbolized by single letters, are 
ordinary matrices, and for states distributed con- 
tinuously are kernels of integral equations. Such 
a representation is also ideally suited for machine 
computation. The formulation also seems much 
more closely related to the intuitive ideas used by 
the experimentalist in studying transport prob- 
lems than some other approaches. Its develop- 
ment makes possible a rather direct road to a 
variety of particular mathematical techniques. 

Particular biological problems studied within 
this framework have been fatty acid transport, 



potassium washout from kidney slices, and 
comitercurrent exchange. 



Fluorescence and Phosphorescence 

A series of isomers of several substituted acet- 
anilides has been pux'ified for the study of the rela- 
tion of their phosphorescent spectra to stracture. 
The compounds will be studied in the aqueous 
propylene glycol glass at 184°K and their spectra 
compared with those obtained in nonaqueous media 
at 184°K and 77°K. 

Several materials (e.g. chlorpromazine, kynu- 
renic acid and fluorescein) that have been shown 
to be phosphorescent on paper at liquid nitrogen 
temperature have been studied. At room tem- 
perature the feeble phosphorescence normally ob- 
tained can be enhanced by the use of infrared or 
electric fields. Subjecting them to such energy 
sources causes these phosphors to emit their 
phosphorescence more rapidly and makes it pos- 
sible to obtain intensities that will allow sensitive 
measurement of such compounds. Various 
glasses, such as vinyl acetate, cellulose acetate 
butyrate and boroglycerine glasses have been 
shown to provide physical conditions that favor 
phosphorescence, but unless the stimulation tech- 
nique can be used to enliance the emission the re- 
sults to date indicate that samples of the order of 
milligrams will be needed for satisfactory 
measurement. 

Freezing Point Determinations 

A two-stage thermoelectric refrigeration system 
has been constructed. The apparatus will rapidly 
cool a sample holder positioned under a micro- 
scope so that samples can be observed while the 
temperature is controlled electrically. Tliis sys- 
tem accommodates 8 samples of one micromilliliter 
each, under oil in holes in the sample holder. The 
current through the thermoelectric elements di- 
rects the flow of heat and rapidly changes the tem- 
perature. The apparatus, now operating, is being 
provided with a servo control system that will 
allow selection of the equilibrium temperature 
while the ice liquid equilibrium is observed in the 
microscope. 



98 



ANNUAL REVIEW OF ESTTRAMUEAL RESEARCH 



LABORATORY OF CARDIOVASCULAR 
PHYSIOLOGY 

Electrolyte Heart Changes During Homeometric 
Autoregulation 

The exhibition of homeometric autoregulation 
by the left ventricle has been amply confirmed in 
the isolated heax-t. It was therefore assiuned that 
an increase in the amornit of tension developed 
per unit of time by the myocardimn must be fol- 
lowed by the development of some biochemical 
rearrangement which provides for an increased 
contractility. We had previously suggested the 
possibility that changes in intracellular potassium 
might play a role in this phenomenon. It has now 
been shown that a net efflux of potassium does 
occur when the total tension developed by the 
myocardium is increased either as a result of in- 
creasing aortic pressure or heart rate. The 
amount of the net efflux is small. However, it 
has been shown that this efflux is comparable to 
that observed when a comparable increase in con- 
tractility is produced by the administration of 
glycosides. 

It will be difficult to establish a cause-effect rela- 
tion between the hemodynamic and the biochemi- 
cal changes observed. However, the magnitude 
of the intrinsic changes in contractility observed 
underscore the importance of continued efforts to 
characterize the biochemical changes. 

The following premises are the basis of a work- 
ing hypothesis : 

A. Homeometricity is observed when myo- 
cardial TTI (the amount of tension developed 
by the heart per unit of time) is increased 
(established) . 

B. The O2 consumption of the heart is de- 
termined by TTI ; when TTI is increased the 
O2 consumption of the heart is increased 
(established). 

C. When the O2 consmnption is increased, 
CO2 production is increased and intracellular 
PCO2 and hydrogen ion concentration are in- 
creased; these are changes which influence 
intracellular potassium (assumed). 

D. A net efflux of potassium occurs when 
TTI is increased (establislied). 

E. When intracellular potassium is low- 



ered myocardial contractility is increased 

(widely considered to be established). 

One valuable piece of information growing out 

of this study is the recognition of the occurrence 

and the magnitude of Bowditch effect in the intact 

heart. 

Isolated Papillary Muscle 

The basic mechanics of heart muscle have been 
explored using the cat papillaiy muscle. By re- 
lating the force the muscle develops to its velocity 
of shortening, two helpfial generalities have been 
clearly demonstrated. 1) Increasing muscle length 
increases tlie maximal force of the muscle but 
does not alter its intrinsic (maximal) velocity. 
2) Inotropic interventions (heart rate or nore- 
pinephrine), at any one muscle length, increase 
intrinsic velocity of the muscle with a variable 
change in force. This increase in intrinsic veloc- 
ity defines inotropism. The active compliances 
of the muscle have been shown to be constant in 
the face of these changes and thus do not con- 
tribute to the observed phenomena. Interrela- 
tions of work and power have also been studied. 
This has yielded information on the influence of 
afterload on performed work and work capacity. 
This type of analysis forms the background with 
which to compare phenomena observed in the in- 
tact heart. 



Afferent Pathways Lifluencing Efferent Auto- 
nomic Discharge 

Carotid Body Hypoxia. 

As previously shown, carotid body hypoxia 
causes a marked bradycardia when respiration is 
controlled. This was reduced, but not abolished 
by vagotomy. The subsequent administration of 
hesamethonium completely abolislied the resjionse, 
thus establishing that the observed bradycardia is 
attributable to sympathetic withdrawal as well as 
increased vagal activity. Further, the contrac- 
tility of the atrium is reduced by carotid body 
hypoxia and varying degrees of heart block are 
frequently observed. The latter responses are 
considered to be due to efferent vagal activity. 

Ventricular fmiction curves showed tliat carotid 



NATIONAL HEART INSTITUTE 



99 



body hypoxia causes a reduction, never an increase, 
of ventricular contractility, indicating a reduction 
of sympathetic discharge to the heart. The reduc- 
tion in heart rate after vagotomy and the reduc- 
tion in ventricular contractility are associated with 
a concomitant increase of total peripheral resist- 
ance. These findings show that hypoxic stimula- 
tion of the carotid bodies causes a dichotomous 
sympathetic response, that is, a reduction of 
sympathetic discharge to the heart and a simul- 
taneous increase of sympathetic discharge to the 
peripheral vasculature. Such a dichotomy has 
not previously been demonstrated. 

Central Nervous System Hypoxia 

Since the response to total body hypoxia differs 
markedly from that observed with carotid body 
hypoxia, it was reasoned that other mechanisms 
miglit be involved. Accordingly, a preparation 
was developed in which differential cerebral 
hypoxia could be induced. A marked mcrease in 
ventricular contractility (as well as peripheral 
resistance and heart rate), such as is observed in 
total body hypoxia, occurred. 

Cardiac Sympathetic Nerve Discharge During 
Carotid Body and Central Nervous System 
Hypoxia 

An electroneurographic correlation with A and 
B above was obtained by means of separate studies 
in which nerve impulses were recorded from the 
lesser cardiac nerve in the cat. Wliereas systemic 
hypoxia caused a marked and persistent increase 
in the sympathetic discharge to the heart even 
after denervation of the peripheral chemorecep- 
tors, isolated carotid hypoxia lessened this imjiulse 
activity. 

Vagal Ajferenf Pathways 

The stimulation of certain afferent vagal fibers 
has been shown to produce a very marked increase 
in left ventricular contractility as demonstrated 
by the shift of the ventricular function curve. 
The possible role of these fibers, with special ref- 
erence to the location and type of receptor they 
serve is the cause of considerable speculation in 
this laboratory at the present time. Tlie presence 



of chemoreceptors on the inflow portion of the 
pulmonary vascular bed is being investigated. 

Ejfect of Carotid Hypotension on Renal Blood 
Flow 

A teclxnique has been developed to meter and/or 
control renal blood flow without perceptible mtei'- 
ference with renal innervation. Studies per- 
formed using this teclmique demonstrate the im- 
portance of reflex influences on renal function. 
The major findings in this study to date are: 

(1) Carotid occlusion is accompanied by an 
increased renal vascular resistance; during 
occlusion renal blood flow may fall as renal 
arterial pressure increases. 

(2) "Wlien the kidney is perfused at a con- 
stant flow, renal arterial pressure increases in- 
dicating that the response is due, at least in 
part, to extrinsic nervous influences and not 
simply to autoregulation. 

(3) In many animals, a given change in 
renal perfusion pressure produced by carotid 
occlusion is accompanied by little or no 
change in renal blood flow, whereas the same 
change in pressure produced by decreasing 
renal inflow resistance produces a substantial 
change in renal blood flow. 

(4) The fall in renal blood flow wliich may 
be observed during carotid occlusion can be 
changed to an increase in blood flow by 
dibenzyline, at a time when dibenzyline itself 
does not modify the resting blood flow. 

( 5 ) Conversely when arterial pressure is in- 
creased, as with stellate ganglion stimulation, 
renal blood flow rises and diuresis occurs. 

Vagal Afferent Pathways Indtocing Reflex Vasodi- 
lation and Hypotension During Myocardial 
Iscliemia 

The hypotension accompanying some instances 
of myocardial infarction has long been thought to 
be due almost exclusively to myocardial incom- 
petence. In a dog liindlimb perfused at constant 
flow, it was demonstrated that acute occlusion of 
the circumflex brancli of the left coronary artery 
reflexes induces a marked peripheral vasodilation. 
Cooling of the vagi abolished this response which 
in turn, reappeared when the vagi were rewarmed. 



100 



AJSnsrUAL REVIEW OF INTRAMUKAL RESEARCH 



Further evidence of sympathetic inhibition under 
these circumstances was obtained by electroneuro- 
graphic tecliniques in the cat; a persistently di- 
minished sympatlietic impulse rate was obtained, 
while the vagi were intact, when local myocardial 
ischemia was induced. 

Dynamics of the Cardiac Cycle 

The Ventricle. RemodynamiG Determinants of 
the Duration of Left Ventricular Systole, 
Ejection and Isovolumic Gontrcuition 

These studies were carried out in a newly de- 
veloped right heart bypass preparation. The fol- 
lowing observations were made : 

1. The effect of increasing stroke volume 
is to prolong ventricular ejection with little 
or no effect on total duration of ventricular 
systole. There is, therefore, a progressive 
shortening of the isovolumic contraction 
period as stroke volume is augmented. 

2. The effect of increasing mean aortic 
pressure is to reduce ventricular ejection time 
with little or no effect on the total duration 
of ventricular systole. There is therefore a 
prolongation of the isovolumic contraction 
period. 

3. The effects of augmenting heart rate are 
to shorten both the duration of ventricular 
ejection and total systole, the former more 
than the latter so that duration of isovolumic 
contraction is decreased. 

4. The independent augmentation of heart 
rate, aortic pressure and stroke volume each a) 
increase the mean rate of ventricular ejection, 
and b) increase the average rate of rise of 
ventricular pressure during isovolumic con- 
traction. 

The data are consonant with the view that in- 
creasing LVED fiber length, increasing heart rate 
and increasing mean aortic pressure each lead to 
a more vigorous ventricular contraction. 

TTie Atrium, 

Study of the participation of the atria in circu- 
latory hemodynamics and regulation has been con- 
tinued with particular reference to the transport 
function of the atria. Evidence was obtained in- 
dicating that the duration of ventricular diastole, 
the vigor of atrial systole and the timing of atrial 



systole are the fundamental factors influencing 
the relationship between mean left atrial pressure 
and left ventricular end diastolic pressure and 
thus the relation between the central pressure head 
which must be overcome by blood returning from 
peripheral vessels and the hemodynamic stimulus 
to the ventricle's force of contraction. Independ- 
ent variation either of mean aortic pressure or 
of stroke volume had little or no influence on this 
relation; thus, changes in heart rate and sympa- 
thetic and vagal tone, because of their influence on 
the vigor of atrial systole and on diastolic time 
appear to be those influences which most impor- 
tantly affect this relation. 

Studies on atrial fibrillation, induced either 
electrically or with aconitine, were initiated in 
the dog with surgically induced heart block. This 
was done so as to be able to ascribe observed 
changes to alterations in atrial activity with the 
ventricle beating at a constant and regular rate. 
The observations obtained thus far suggest that, 
as might be expected, the occurrence of atrial fibril- 
lation is most important at the higher ventricular 
rates. Specifically above a ventricular rate of 
120/min, inducing atrial fibrillation produces a 
rise in mean left atrial pressure for any given left 
ventricular end diastolic pressure. Further, it has 
been observed that, when atrial fibrillation is in- 
duced, mean aortic pressure and cardiac output 
decline even when left ventricular end diastolic 
pressure is maintained. These data appear to 
confirm our previous findings that a properly 
placed and vigorous atrial systole contributes to 
mitral valve closure as well as to ventricular filling. 
The atrium can be considered to perfonn in rela- 
tion to the ventricle those functions that a booster 
pump supplies to a primary power pump in me- 
chanical systems. 

Electrophysiological Studies 

Increased synchronicity of ventricular contrac- 
tion may yield a more forceful contraction from 
any given ventricular end diastolic pressure and 
fiber length. Electrophysiological studies with 
special reference to timing have been initiated to 
investigate this possibility. These studies are 
aimed primarily at determining whether an inter- 
vention such as sj'mpathetic stimulation produces 
a more synchronous ventricular systole. 



NATIONAL HEART INSTITTJTE 



101 



The evidence obtained thus far indicates that, 
in the vagotomized animal and in the animal after 
ganglionic blockade, the effect of increasing heart 
rate is to prolong A-V conduction time whereas 
stellate stimulation and catechol amine infusions 
shorten A-V conduction times. Vagal stimula- 
tion prolongs A-V conduction time. Suggestive 
evidence was obtained indicating that the aug- 
mented heart rate in the animal after ganglionic 
blockade prolongs ventricular activation time 
(QES duration) whereas a similar increase in rate 
produced by stimulation of the cardiac sympathe- 
tic nerves reduces ventricular activation time at 
any given heart rate. These data lend ECG sup- 
port to those micro-electrode studies of similar 
effects of heart rate, sympathetic and parasym- 
pathetic influence on A-V conduction time. 

The usual clinical ECG experience would sug- 
gest that increasing heart rate is associated with 
a shortening of the P-E interval. Our data in- 
dicate that the primary effect of increasing rate 
is to prolong the P-R interval and that the short- 
ening which occurs clinically with increasing rate 
is, therefore, related to the associated increase of 
sympathetic tone. Finally these experiments sug- 
gest and hemodynamic studies of the relation of 
ventricular and atrial dynamics and the effects 
thereon of rate, vagal and sympathetic tone should 
include data concerning the time relationships 
between atrial and ventricular events and the 
activation time of the ventricular myocardium. 

Myocardial Catechol Amines 

Since the cardio-dynamic effects of cardiac 
sympathetic nerve stimulation are related to the 
release of myocardial catechol amines, experi- 
ments were undertaken to study the myocardial 
production and/or utilization of these amines. 
It was found that stimulation of the cardiac sym- 
pathetic efferent nerves was accompanied by a 
release of catechol amines into coronary blood. 
This release was a function of stimulation in- 
tensity and occurred even when stroke volume 
and coronary outflow were maintained constant. 
Comparison between the ethylenediamine and the 
trihydroxyindole methods for the analysis of 
plasma catechol amines showed both quantitative 
and qualitative differences; only the results with 
the latter method showed a consistent relationship 
to the hemodynamic changes observed during car- 



diac sympathetic stimulation. The acutely sym- 
pathectomized heart was found to extract catechol 
amines from coronary blood in the absence of nerve 
stimulation. The patterns of extraction observed 
indicated that the quantity of catechol amines in 
the myocardium modify the extent of extraction 
in the unstimualted state as well as the extent of 
release during stimulation. Continued stimula- 
tion of the cardiac sympathetics was accom- 
panied by at least a partial depletion of the myo- 
cardial catechol amine stores. These stores ap- 
peared to be repleted by circulating catechol 
amines, since the response to any given intensity 
of stimulation was potentiated by a prior nore- 
pinephrine infusion. Dichloroisoproterenol pre- 
vented neither the myocardial extraction of 
catechol amines in the unstimulated state nor the 
release of catechol amines during sympathetic 
stimulation; it did lessen or abolish the cardi- 
odynamic effects of sympathetic stimulation. 
Data were also obtained which indicated that a 
direct coronary vasoconstriction is one of the con- 
sequences of cardiac sympathetic nerve stimula- 
tion, the vasodilatation usually observed being 
due, at least in part, to overriding metabolic 
factors. 

Having established the characteristics which 
appeared to modify the release and extraction of 
myocardial catechol amines during direct nerve 
stimulation and in the acutely sympathectomized 
heart, experiments were undertaken to determine 
whether, during carotid sinus hypotension when 
myocardial contractility is reflexly increased, the 
release of myocardial catechol amines also oc- 
curred. These experiments showed that carotid 
sinus hypotension can be accompanied by the re- 
lease of catechol amines and that the release is 
prevented by bilateral stellectomy. This reflex 
release of catechol amines appears to be inde- 
pendent of changes in cardiac output, heart rate, 
coronary blood flow changes and changes in 
arterial blood pressure. 

Experiments were also done to determine the 
effects of bretylium on the myocardial release and 
uptake of catechol amines. The initial response 
to the injection of bretylium tosylate was a release 
of myocardial catechol amines. This release ap- 
peared to occur from myocardial stores since it 
was observed after the injection of dichloroiso- 
proterenol. After the initial myocardial release 



I 



102 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



of catechol amines, bretylium blocked the release 
of catechols associated with cardiac sympathetic 
nerve stimulation. The failure of nerve stimula- 
tion to release catechol amines after bretylium 
was not due to depletion resulting from the initial 
release, since tyramine still caused a release of 
catechols from the heart in the presence of bretyl- 
ium. Although bretylium prevented the release 
of catechol amines associated with cardiac nerve 
stimulation it does not appear to interfere with the 
myocardial extraction of catechol amines even in 
the presence of dichloroisoproterenol. Thus the 
antihypertensive effect of bretylium appears to be 
related to blocking of sympathetic axon conduc- 
tion. 

Vasculature of Resting Musculo-cutaneous Areas 
During Exercise 

By metering blood flow in resting areas as well 
as in those in which simulated exercise was in- 
duced, it was ascertained that the vasoconstric- 
tion that occurs in the resting areas is preceded 
by a substantial vasodilation. This is not blocked 
by atropine but is abolished either by local sym- 
pathectomy or TEAC indicating a brief initial 
diminution in sympathetic tone in the resting area 
with the onset of exercise. The cholinergic 
vasodilator system of Uvnas and Folkow is ap- 
parently not involved. The secondary vasocon- 
striction in the resting area was abolished by 
TEAC. 

The possible role of the initial vasodilation in 
resting areas in facilitating the transition from 
rest to exercise is not at present understood. 

The Kallikrein System 

Studies on this hypotensive system have pro- 
vided data to suggest that the inborn biochemical 
lesion in hereditary angioneurotic edema is due to 
a deficiency of a serum inhibitor of plasma kalli- 
krein. Two biologically active polypeptides liave 
been isolated from the incubation of human uri- 
nary kallikrein with acid-treated human plasma 
and the structure of these kallidins has been deter- 
mined. Methods have been devised for the j^rep- 
aration of esentially homogeneous human urinary 
and human pancreatic kallikrein. Comparison of 
these proteinases with pure hog pancreatic kalli- 



krein by electrophoresis on cyanagum gel has re- 
vealed that each hypotensive enzyme has its own 
characteristic mobility. Eabbit antibody to crude 
and pai'tially purified human urinary and human 
pancx'eatic kallikreins has been shown to cross 
react, but antibody to human urinaiy kallikrein 
failed to precipitate in agar gel and to inliibit the 
vasodilator activity of hog pancreatic or dog uri- 
nary kallikreins. 

The possibility was investigated that the vaso- 
dilation accompanying skeletal muscle exercise is 
due to activation of the kallikrein-kallidin system. 
These experiments revealed tliat the lymph from 
most dogs during exercise gave only a barely de- 
tectable level of kallikrein as measured on tlie 
uterine strip. 



LABORATORY OF KIDNEY AND ELECTRO- 
LYTE METABOLISM 

The research activities of the Laboratoiy of 
Kidney and Electrolyte Metabolism may be 
divided into several broad categories. These will 
be discussed separately. 

Renal Physiology 

Micropuncture Studies 

In recent years the reapplication of the micro- 
puncture technique of Eichards et al. has provided 
significant new information concerning the site 
and mode of transfer of water and electrolytes 
across the tubular epithelia in the amphibia as 
well as in cei'tain small rodents. No comparable 
studies have been completed in the dog. Eenal 
function in this species appears to resemble that 
in man more closely than m those species in which 
micropunctui'e data are available. Preliminary 
studies in this laboratory have establislied tlie 
feasibility of applying the micropuncture tecli- 
nique to the dog. An elegant system has been de- 
veloped in which micropuncture data may be 
correlated with those obtained during clearance 
studies. It has been shown that reabsoi-ption in 
the proximal tubule is accomplished by abstrac- 
tion of isosmotic fluid. The ratio of the os- 
molality of tubular fluid to plasma remained 1.00 
throughout the accessible portion of the proximal 
nephron during both hydropenia and \vat(M- 



NATIONAL HEART INSTITUTE 



103 



diuresis. It is of interest that distal nephrons 
have not as yet been visualized in the accessible 
portion of the outer cortex. It is proposed to 
examine younger animals in which the proximal 
tubules are less elongated in an effort to determine 
whether the distal nephron will be accessible for 
puncture under these circumstances. Future 
studies are to involve measurements of the poten- 
tial difference across the nephron, the in situ pH, 
etc. 

Studies of acidification in the proximal nephron 
of the Necturas, initiated some time ago, continue. 
The purpose and difficulties of these experiments 
have been detailed in the jsrevious report. In 
general, it is hoped to establish whether or not 
acidification is accomplished by direct reabsoi-p- 
tion of bicarbonate or by secretion of hydrogen 
ions in exchange for sodium, as is generally ac- 
cepted. The latter view, which involves conver- 
sion of luminal sodium bicarbonate to carbonic 
acid, requires that the rate of dehydration of car- 
bonic acid be equal to the rate of bicarbonate re- 
absorption. In the absence of carbonic anhydrase 
the non-catalyzed dehydration of H2CO3 is rate 
limiting. In order for it to proceed at a rate 
equivalent to that of maximal bicarbonate re- 
absorption, the in situ pH must be approximately 
1 pH unit less than that of plasma. In contrast if 
carbonic aniiydrase is present in the luminal fluid 
or on the membrane the dehydration process will 
be virtually instantaneous and the in situ pH 
should approximate that of plasma. Carbonic 
anhydrase inhibitors should decrease rather than 
increase the pH under these circumstances. 
Though these considerations are amenable to ex- 
perimental verification, the major deterrent has 
been the unavailability of pH sensitive micro- 
electrodes. In recent months some of the diffi- 
culties have been eliminated and a pH sensitive 
microelectrode has been successfully prepared. 
In preliminary studies it appears that the in situ 
pH in Nectunis does not differ from that of 
plasma. No studies utilizing carbonic anhydrase 
inhibitors have been completed. However, as an 
important check on the validity of the technique, 
the transtubular and transcellular potential differ- 
ences have been determined. The values observed 
were similar to those reported by others. 



Garionic Anhydrase in Rat Renal Cortex 

Methods for autoradiographic localization of 
carbonic anhydrase in renal tissue are being devel- 
oped in collaboration with the Department of 
Anatomy of Georgetown University. It is hoped 
to determine the cellular localization of carbonic 
anhydrase as well as its presence or absence in the 
luminal membrane. The experimental approach 
is dependent on and takes advantage of the known 
high binding constant of acetazoleamide, a car- 
bonic anhydrase inhibitor, and the enzyme car- 
bonic anhydrase. Theoretically, it should be pos- 
sible to localize autoradiographically the site of 
the enzyme-inhibitor complex if sufficient labeled 
acetazoleamide (H^) is presented to the intact 
renal cortex. 



Measurement of Medullary Blood Flow 

In view of the important role of the counter- 
current vascular system in the medullaiy and 
papillary portion of the kidney, in the concentrat- 
ing and diluting process, studies have been di- 
rected at developing a technique for the estima- 
tion and evaluation of renal medullary blood flow 
in the intact dog. The purpose of these studies as 
well as the pertinent background information 
were discussed in detail in the last report. It is 
now generally accepted that the vascular counter- 
current system is largely responsible for the main- 
tenance of the osmotic gradient in the interstitial 
spaces of the kidney. In recent months it has 
been possible to estimate effective flow indirectly, 
by measuring the rate of accumulation of diffusi- 
ble gas, in this instance, molecular hydrogen, by 
both cortex and medulla. A technique requiring 
imjDlantation of a platinum electrode responsive 
to hydrogen tension has been devised. Hydrogen 
is administered intratracheally to the dog and 
effective flow to the medulla and cortex are esti- 
mated. In acute studies hydrogen is veiy effec- 
tively excluded from the medulla in antidiuresis, 
and its rate of entry is considerably increased 
when urine flow is elevated by administration of 
osmotic diuresis. These studies are now being 
extended to dogs in wliich electrodes for chronic 
studies have been implanted in both cortex and 
medulla. 



104 



AJSnsnCJAL REVIEW OF rNTRAMURAL RESEARCH 



E-ffect of Diuretic Agents in Vasopressin-Be- 
sistant Diabetes Insipidiis 

Studies concerning the influence of diuretic 
agents on urinary dilution in patients with vaso- 
pressin resistant diabetes insipidus have been 
completed. In the previous report it was noted 
that chlorotliiazide, a diuretic agent, which inter- 
feres with sodium chloride reabsorption in the 
diluting segment, results in an expected rise in 
urine osmolality and a paradoxical fall in urine 
flow. The latter effect contrasts strikingly with 
that observed in normal individuals in whom an 
increase in urine flow is uniformly observed. It 
was suggested that the fall in urine flow may be 
conditioned by the resultant sodium depletion. 
The latter may provide the basis for a diminution 
in volume flow throughout the nephron either as 
a result of a decrease in filtration rate or a com- 
pensatory increase in proximal reabsorption. If 
sodium depletion per se is responsible for the de- 
crease in urine flow, then antidiuresis should also 
be achieved with mercurial diuretics. This pre- 
diction proved correct in two patients exammed. 

Phosphate /Secretion in the Dog 

Investigations designed to attempt to demon- 
strate net phosphate secretion in the dog have been 
completed. Although such secretion has been re- 
ported by others, the evidence has not been con- 
vincing. Repeated attempts, utilizing all of the 
many experimental manipulations reported to re- 
sult in phosphate secretion, have in our hands pro- 
vided no evidence of this phenomenon in the dog. 
The present studies differ from earlier ones in an 
important respect. Steady state conditions inso- 
far as possible were maintained throughout; that 
is filtration rate was relatively constant as was 
plasma phosphate concentration. 

Electrolyte and Water Transport Across Biolog- 
ical Membranes 

Renal Cortical Slices and Tubule Suspensions 

For a number of years this laboratoi-j' lias been 
engaged in a study of the transfer of K^- into and 
out of renal cortical slices. Although the results 
of these studies have generally been confirmatory 
of others it was recognized that the technique may 
provide little information concerning the trans- 



membrane flux of K^= across the pertinent cell 
borders. The interposition of an interstitial space 
through which isotope must diffuse complicates 
interpretation of flux data. In general, studies 
such as these merelj' afford information concern- 
ing influx and efflux of isotope into and out of the 
whole tissue. In preliminary studies utilizing a 
constant flow teclinique m which isotope is jjassed 
continuously over a single slice immersed in the 
well of a scintillation counter, it was possible to 
establish conditions which provided reproducible 
data under a variety of circumstances. The tech- 
nique was described in detail in an earlier report. 
Utilizing this method, it was observed that the 
cardiotonic steroid, strophanthidin, diminished 
the uptake of Iv*^ into the slice, an effect analogous 
to that observed in other tissues including the red 
cell ; however, in contrast to the red cell the wash- 
out of K''= was markedly accelerated. In view of 
the complications afforded by the presence of an 
interstitial space it was considered that the latter 
effect, the augmented K^- eiflux, was an experimen- 
tal artifact and that efflux across the cell membrane 
was either unchanged or diminished. In order 
to determine transmembrane fluxes with some de- 
gree of certainty it was necessary to eliminate the 
interstitial space. A technique for the prepara- 
tion of a suspension of isolated proximal tubule 
segments was devised. The technique involves 
the dissolution of the interstitial space by perfu- 
sion of the intact kidney with collagenase (a pro- 
teolytic enzyme) and the subsequent collection of 
the short lengths of proximal tiibule. The cell 
suspensions proved viable as demonstrated by nor- 
mal respiration (oxygen consumption greater than 
in comparably studied slices), accumulation of 
PAH to a degree gi-eater than \n slices, and 
maintenance of normal tissue electrolj'te composi- 
tion. The effect of strophanthidin on K"- flux 
in this preparation supported the initial conten- 
tion that augmented flux in the slice was an ex- 
periment artifact. As in the i-ed cell and cortical 
slices, the drug interferes with the uptake of K'" 
but in contrast to the results in slices efflux of K'= 
is actually diminished. This last is similar to that 
observed in the red cell. The results cast con- 
siderable doubt on the validity of kinetic analyses 
of transport phenomena in intact cortical slices, 
and it is proposed to reexamine these in the tubule 
suspension system. 



NATIONAL HEART INSTITUTE 



105 



Sodium, and Potassiwm Dependent AT Pose in the 
Renal Cortex 
A limited characterization of a sodiiun- 
potassium dependent ATPase in dog renal cortical 
tissue has been completed. The enzyme, which 
requires sodium and potassium for activation, has 
been observed in other tissues including crab 
nerve, red cells and guinea pig kidney. Since its 
activity has been shown to correlate with cation 
transport in red cells, it is presumed to be involved 
directly in the carrier mediated transport process. 
It is present in high, though variable concentra- 
tion, in dog renal cortex. Homogenates require 
appropriate concentrations of sodium, potassium, 
magnesium and buffer for maximal activation. 
Of interest is the observation that the activity of 
the enzyme is depressed by as much as 75% fol- 
lowing incubation with the cardiotonic steroid, 
strophanthidin. This may be cited as indirect 
evidence in support of the view that the enzyme 
is intimately involved in the cation transport 
process, since strophanthidin also interferes with 
active transport in this tissue. Aldosterone, the 
adrenal steroid which is thought to stimulate ca- 
tion transport, had no effect on the activity of the 
enzyme, whereas the addition of either organic 
mercury or calcium effectively inhibited the 
activity. 

Cation Transport in Red Cells 
Alterations in Lipid Content. Studies of the 
mechanism of active cation transport m intact red 
cells and red cell ghosts comprise a significant 
segment of this laboratory's research activity. 
Attention has been focused on the influence of the 
lipid composition of the red cell membrane on 
cation transport. Three groups of red cells were 
studied — (a) rat red cells obtained from fatty 
acid deficient animals in which the fatty acid 
content of the cells was markedly altered; (b) 
human red cells from patients with acanthocytosis 
in whom the fatty acid content of the cells is 
altered in a fashion similar to (a) above; and (c) 
cells from vitamin E deficient rats. Despite sig- 
nificant changes in the lipid structure of the cell 
membrane the transport of sodium and potassium 
was unaffected. 

TiiANSPOET in Eat Red Cells. In the course of 
these studies a number of interesting observations 
were made relative to the kinetics of electrolyte 



transport in normal rat red cells. These proved 
of greater interest than the original purpose of 
the studies and may provide a tool for the exami- 
nation of carrier mediated transport. It was 
noted that rat red cells contrast strikingly with 
those from other species studied. The rat pos- 
sesses a sodium-potassium linked exchange pump, 
however the curve relating tlie activation of 
sodium outflux by external potassium does not 
resemble the classical Michaelis-Menten curve but 
rather is described by a sigmoid relationship ; that 
is, negligible sodium transport occurs until the 
concentration of potassium is elevated above a 
critical level. Similar considerations apply to the 
curve relating potassium influx to external potas- 
sium concentration. It is also of interest that 
strophanthidin, a potent inhibitor of transport in 
himian red cells, is relatively ineffective in this 
species, whereas scillaren is extraordinarily active. 
Furthermore, it was noted that exchange diffusion 
of sodium is inhibited by concentrations of scil- 
laren wliich are without influence on active 
sodium-potassium exchange. An elevation of the 
external potassium concentration resulted in the 
reappearance of exchange diffusion. Although 
these studies are still in a preliminary stage, it may 
be reasonable to consider that active transport and 
exchange diffusion of sodium, though both carrier 
mediated, occur at different sites on the membrane. 

Influence of Divalent Cations on the Peemea- 
BiLiTT op the Eed Cell TO Na AND K. Ill associa- 
tion with the above studies in intensive investiga- 
tion of the effect of divalent cations and metabolic 
alterations on permeability to cations of liuman red 
cells had been undertaken. It has been known that 
the addition of calcium results in a marked in- 
crease in the passive permeability to potassimn in 
red cells exposed to iodoacetate (lAA) and adeno- 
sine. This effect, which requires all three sub- 
stances, served as a basis of the following studies. 
Normal and substrate depleted human red cells 
were examined with respect to the effects of (a) 
calcium, (b) lAA (and other inhibitors) and (c) 
adenosine (or other substrates) on the movement 
of potassium and sodium. It was noted in normal 
cells that the augmentation of potassium perme- 
ability required the presence of either calcium or 
strontium, that lAA could be replaced by iodoac- 
etamide, NEM or BAL, with similar results, and 



106 



ANTSfXJAL REVIEW OF INTRAMURAL RESEARCH 



that inosine was the only compound which could 
substitute for adenosine. In contrast to the above 
effects in normal cells, energy depleted cells were 
responsive to calcium alone (i.e. without inhibitor 
or substrate). Furthermore, although a greater 
effect was obtained in partially depleted cells by 
addition of lAA and adenosme, adenosine exerted 
no effect when cells were depleted for 25 hours or 
longer. These cells respond maximally to calcium 
alone. Also preexposure of cells to adenosine or 
inosine prevented the subsequent action of cal- 
cium, lAA and adenosine on potassium permeabil- 
ity. Although final conclusions are not yet war- 
ranted it is sugegsted that potassium permeability 
is regulated both by calcium and a metabolic prod- 
uct which can be removed by a reaction involving 
adenosine (as limited by lAA) or by depletion of 
the cell. Eemoval of the metabolite predisposes 
the membrane to the action of calcium which is 
ineffective alone in non-depleted cells, but is ef- 
fective in depleted cells even without the addition 
of lAA and adenosine. None of the above ma- 
nipulations was capable of altering the passive 
permeability of the red cell to sodium. How- 
ever, the inhibition of active sodium and potas- 
sium transport which occurs following addition 
of glycolytic inhibitors, such as lAA, is accen- 
tuated by the simultaneous addition of adenosine. 
Furthermore adenosine inhibits exchange diffu- 
sion in cells exposed to lAA. These latter observa- 
tions support the view that an imknown product 
of metabolism is important in the maintenance 
of the integrity of the membrane with respect to 
carrier mediated transport as well as passive 
diffusion. 

Water Movement Across the Toad Bladder 

Vasopressin. The antidiuretic hormone, vaso- 
pressin, reduces urine flow and permits the excre- 
tion of a concentrated urine in the intact animal, 
by increasing the permeability of the distal neph- 
ron to water. The increase in permeability is 
thought to be accomplished by hormone-induced 
enlargement of aqueous channels (pores) in both 
the distal convolution and collecting duct. This 
view of the action of vasopressin is based on stud- 
ies of water movement across the epithelial 
structure of frog and toad, skin and bladder. In 
all of these, addition of vasopressin to the serosal 
(blood surface) of the isolated membranes ac- 



celerates the osmotic flow of water across the 
structure. In association with this, net movement 
of sodimn is also accelerated, as evidenced by an 
increase in the so-called short-circuit current. 
Little information is available concerning the 
metabolic effect of the hormone and it has been 
suggested that binding of the hormone by its SS 
bridge to SH groups on the membrane, and a 
subsequent interchange reaction, springs open 
pores in a mechanical fashion without the inter- 
position of any known metabolic energj' sources. 
The latter thesis is based on the observation that 
vasopressin binds both to renal tissue and toad 
bladder and that prevention of binding by either 
acidification of the bathing medium or the addi- 
tion of sulfhydryl inhibitors prevents the permea- 
bility effect of the hormone. An alternative thesis 
developed in this laboratoi'y involves the inter- 
mediacy of adenosine 3'5 phosphoric acid, a cyclic 
nucleotide (cyclic AMP) . It is known that effects 
similar to those of ACTH in the adrenal and 
glucagon (or epinephrine) in the liver can be 
produced with vasopressin. ACTH and glucagon 
have in common the ability to stimulate the forma- 
tion and accumulation of cyclic AjVIP in their 
respective receptor tissue. The nucleotide in both 
tissues accelerates the conversion of inactive to 
active phosphorylase, the enzyme which converts 
glycogen to glucose-1-phosphate, and is considered 
to be an integral factor in the development of the 
physiologically recognizable effects of the hor- 
mone. On the basis of these observations it was 
considered that vasopressin may simulate ACTH 
in the adrenal and glucagon in the liver by virtue 
of a stimulatory effect on cj'clic AMP production. 
More pertinent to the present discussion was the 
assumption that cyclic AMP may also be an inter- 
mediate in the action of vasopressin in both the 
kidney and toad bladder. It was suggested that 
the hormone accelerates the conversion of ATP 
to cyclic AMP in these tissues and that the latter 
compound in some as yet undefined manner di- 
rectly or indirectly increases the permeability of 
the membrane to water. It is of significance in 
this regard that theophylline, a methyl xanthine, 
prevents the degradation of cyclic AMP in other 
tissues and thereby conceivably could magnify the 
effect of endogenously formed cyclic AMP. 

Evidence in favor of this view of the action of 
vasopressin was developed utilizing the toad blad- 



NATIONAL HEART INSTITUTE 



107 



der sac as an experimental model. Addition of 
vasopressin to the outer surface of the bladder sac 
markedly accelerates the osmotic flow of water 
across the membrane and stimulates active sodium 
transport. No other substances have been known 
to exert this unique efl'ect. However, the addition 
of cyclic AMP to the serosal surface results in an 
immediate increase in water movement indistin- 
guishable from that due to vasopressin. Short 
circuit current is also increased. Theophylline, 
which as noted earlier, interferes with the break- 
down of cyclic AMP, exerted similar efl'ects on 
water movement and sodiiun transport, as did 
vasopressin and cyclic AMP. N-ethylmaleimide, 
a sulfhydryl inhibitor, which prevents the action 
of vasopressin also interferes with the action of 
cyclic AMP and theophylline. Acidification of 
the bathing medium which interferes with the ac- 
tion of vasopressin in toad bladder also limits the 
effect of theophylline on water movement but does 
not inhibit the effect of cyclic AMP. Since acidi- 
fication interferes with the conversion of ATP to 
cyclic AMP in other tissues a similar effect in the 
toad bladder could account for these results. In 
association with the above studies it has been pos- 
sible to demonstrate an increase m phosphorylase 
activity in toad bladder following incubation with 
vasopressin. Though this is presumed to be medi- 
ated by an increase in the concentration of cyclic 
AMP, direct measurements of the cyclic nucleotide 
have not yet proved feasible. The latter project 
is being actively pursued in collaboration with the 
Department of Pharmacology of Western Reserve 
University. 

Estimation of Vasopressin in Biological Fluids. 
Interest in the metabolism and action of vasopres- 
sin has provided a stimidus for initiating an in- 
vestigation of the feasibility of a modified double 
isotope derivative method for the quantitative es- 
timation of vasopressin in biological fluids. The 
method was originally developed for the measure- 
ment of aldosterone. At present vasopressin can 
be estimated only by means of a relatively non- 
specific bioassay method. To date a technique for 
the isolation of pure arginine vasopressin from 
crude pituitary powder has been developed. Vaso- 
pressin has been acetylated successfully with non- 
labelled acetic anhydride prior to adding the 
labelled compounds for measurement. The stoi- 
chiometry of the process is being examined at 
present. 



Effect of Soltjte Concentration on Net Water 
Mo^TEJiENT Across Toad Bladder. The labora- 
tory has also been engaged in a detailed examina- 
tion of the characteristics of osmotic flow of water 
across the toad bladder utilizing the toad sac tech- 
nique. As indicated above, vasopressin uniformly 
augments the osmotic flow of water across this tis- 
sue. The imposition of a 220 milliosmolal gradi- 
ent (serosal concentration greater than mucosal) 
results in a considerably greater net flow of water 
out of the sac than when the gradient is reversed 
and net flow is directed inward. This rectification 
phenomenon occurs over a significant range of 
osmolalities. Hypotonicity of the serosal bathing 
solution (osmolality below 220) exerts an inde- 
pendent effect since it decreases the permeability 
to H2O in both directions without altering the 
rectification phenomenon. Hypertonicity of the 
serosa also appears to lower the permeability some- 
what. In contrast hypertonicity of the mucosal 
bathing solution significantly lowers the permea- 
bility to water on that surface. These observa- 
tions are being reexamined utilizing tritiated 
water in an effort to determine the changes in the 
unidirectional fluxes of water across both surfaces 
of the membrane as affected by variations in the 
imposed osmotic gradient. Similar studies are 
also being performed using labelled urea and thus 
far appear to suggest that the simple pore hy- 
pothesis involving bulk flow of water across the 
membrane, discussed in an earlier I'eport, is in- 
adequate to account for all of the results. 

Water Movement Across an Artificial Liquid 
Membrane. The validity of the pore hypothesis 
has also been questioned in experiments utilizing 
an artificial membrane. The technique involves 
the use of a non-aqueous liquid membi-ane, mesityl 
oxide, separating two aqueous phases of differing 
osmolalitities. Preliminary results were discussed 
in the pi-evious report. The studies were desigiied 
to test the existing hypothesis which states that 
a discrepancy between the ratio of the water ac- 
tivities on two sides of the membrane and the 
ratio of the unidirectional fluxes of water across 
the membrane are explicable on the basis of bulk 
flow of solvent through aqueous channel or pores. 
As noted in the previous report, the non-iDorous 
mesityl oxide membrane exhibited those charac- 
teristics previously ascribed to a porous biological 
structure. Thus, net flow of water along an os- 
motic gradient was greater than that predicted by 



108 



ANNUAL REVIEW OF INTEAMURAL RESEARCH 



simple diffusion and the activities of water on two 
sides of the membrane. Further studies have con- 
firmed and extended these original observations. 
Furthermore, solvent drag of urea, an augmenta- 
tion in the unidirectional movement of the solute 
(initially equally distributed in both aqueous 
phases), in the direction of net water flow was 
observed ia the mesityl oxide system. So-called 
solvent drag as defined above, has also been cited 
as unequivocal evidence for the presence of pores 
in biological membranes. It appears likely that 
the phenomena observed in the mesityl oxide sys- 
tem, which clearly is unrelated to pores or bulk 
flow of solute, is in part due to a differential par- 
titioning of water in the mesityl oxide at the two 
interfaces separating it from the aqueous solution. 
Although the partitioning is greater in the bound- 
ary separating the phase of higher water activity 
from the mesityl oxide, the relationship between 
the activity of water and its partitioning is non- 
linear as are the unidirectional fluxes of tritiated 
water into the liquid membrane. This non- 
linearity may play a key role in understanding 
the basis for the discrepancies between the flux 
and activity ratios. Because of the non-linearity 
the unidirectional flux of water is greater from 
the more dilute solution into the mesityl oxide than 
from the more concentrated solution. On the 
basis of these studies it has been concluded that 
the concept of bulk flow and solvent drag as de- 
rived from the flux ratio- activity ratio discrepancy 
proposed by Ussing, are not necessarily evidence 
of the presence of pores. It is probable that the 
considerations developed above should also be ap- 
plied to biological membranes. 

Aldosterone 

Aldosterone Stimulating Hormone 

One of the more significant advances reported 
from this laboratory in the past year was the un- 
equivocal demonstration that the kidney secretes 
a substance capable of stimulating the release of 
aldosterone from the adrenals. The original 
studies have now been confirmed and extended. 
Though unrecognized at the time, it is now ap- 
parent that so-called ASH (adrenal stimulating 
hormone) is renin, presumably formed in the 
juxtaglomerula apparatus in the renal cortex. In- 
direct evidence has also been obtained supporting 



the view that ASH is formed in all experimental 
situations associate-d with an increase in aldos- 
terone secretion. Thus, nephretomy reduces 
aldosterone secretion in dogs in which the secre- 
tory rate was initially increased by acute blood 
loss, chronic sodium dejjletion or chronic thoracic 
vena caval constriction. 

Benin- Angiotensin System 

Eecently studies have been completed wliich 
have further clarified the role of the renin-angi- 
otensin system in experimental renal hypertension 
insofar as it relates to enhanced aldosterone secre- 
tion. The injection of renin into hypophysectom- 
ized-nephrectomized dogs increases the rates of 
secretion of aldosterone, corticosterone and Por- 
ter-Silber chromogens. Similar effects are also 
noted following injection of synthetic angiotensin 
II. It is also possible to administer a dose of 
angiotensin which, though incapable of elevating 
arterial pressure, stimulates the secretion of 
aldosterone without a physiologically significant 
change in either corticosterone or Porter-Silber 
chromogen release. Further support in favor of 
the thesis that renin represents ASH was the ob- 
servation that the renin content of kidneys from 
dogs with malignant experimental renal hyper- 
tension is increased 10 fold above normal. These 
animals secrete abnormally increased amounts of 
aldosterone. In contrast the renin content of kid- 
neys from dogs with the benign form of the dis- 
ease is only double that of normals and these 
animals secrete aldosterone at relatively normal 
rates. 

Tlie Effect of Angiotensin on Vascular Resistance 

In view of the evidence suggesting that an- 
giotensm II may be a trophic hormone it is prob- 
able that a homeostatic mechanism is present 
which regulates the incidental action of angioten- 
sin on the vascular resistance of the arterial tree. 
This is consistent with the observations that cer- 
tain patients with enhanced aldosterone secretion 
in whom angiotensin II is presmnably increased 
are not hypertensive, for example, cirrhotics, 
nephrotics, etc. It has been suggested that the 
vascular tree in these subjects is less resi^onsive to 
the renin-angiotensin system, thereby accounting 
for the absence of hypertension. It has also been 
observed in this laboratoiy that the blood pressure 



NATIONAL HEART INSTITUTE 



109 



response to angiotensin II in animals with caval 
constriction is considerably less than that in nor- 
mals. In an effort to obtain direct evidence in 
this regard the contractile response to angiotensin 
of arterial strips from normal dogs is being com- 
pared to that of animals with hj'peraldosteronism 
and no hypertension. 

Formation of ASH 

The chemical nature of ASH has been studied 
in collaboration with the Laboratory of Chemical 
Pharmacology. Crude saline extracts of dog kid- 
ney were fractionated by heat, dialysis, and am- 
monium sulfate precipitation. Various fractions 
were tested biologically in dogs for evidence of 
ASH and pressor activity. It is notable that only 
those fractions separated in a mamier similar to 
that required for the isolation of renin from renal 
tissue possessed steroidogenic and pressor activity. 

Utilizing standard procedures for the separation 
of renin from kidney, it was also noted that tissue 
from animals with thoracic caval constriction 
contained considerably more renin than that from 
normal animals. Furthermore, the juxtaglom- 
erular cells in the former animals were hyper- 
granulated and hyperplastic, ancillary evidence 
in favor of increased renin production. Prelim- 
inary studies have also indicated that the stimulus 
for renin secretion may be related to a fall in 
arterial pressirre and renal blood flow. Thus 
supra-adrenal aorta constriction of hypophysec- 
tomized dogs with reduced arterial pressure and 
renal blood flow resulted in a substantial increase 
in aldosterone secretion. 

Humoral Sensitization of Renal Tubule Cells to 
Aldosterone 

On the basis of studies reported last year it was 
suggested that caval constriction may sensitize the 
renal tubules to aldosterone. The bilaterally 
adrenalectomized and nephrectomized dog with 
one kidney transplanted to the neck, responded to 
caval constriction and DOCA administration by 
retaining sodium. Wlien the caval ligature was 
released, sodiiun was no longer retained despite 
continuing desoxycorticosterone administration. 
These findings suggest that a humoral factor is 
responsible for sensitization of the tubule cells to 
salt-retaining hormone. 



Dlsaffearance of Aldosterone From Plasma 

As reported last year chronic hepatic, venous 
constriction markedly prolongs the half-time 
(Ti/^) for disappearance of injected d-1 or d- 
aldosterone. In nomial animals the T14 varied 
from 20 to 34 minutes, whereas in dogs with 
thoracic vena caval constriction the Ti^ for dis- 
appearance was 43 to 128 minutes. Hepatectomy 
in the former group resulted in a flattening of the 
plasma disappearance curve, indicative of the role 
of the liver in the metabolism of the steroid. 
Kinetic analyses of the data in association with the 
computer division of the N.I.H. supported the 
origmal contention that disappearance of aldos- 
terone from plasma is largely attributable to deg- 
radation by the liver. Despite this, it was con- 
cluded that hypersecretion of aldosterone is of 
more importance in the development of hyper- 
aldosteronemia than decreased turnover in the 
liver in dogs with hepatic venous congestion. 

Effect of Cardiac Glycosides on Aldosterone 
Secretion 

Studies of the acute effects of cardiac glycosides 
on aldosterone secretion in dogs with hyperaldos- 
teronism secondary to chronic right heart failure 
have been completed. In these, improvement in 
hemodynamics following digitalization was uni- 
formly associated with a decrease in aldosterone 
secretion. In animals in which digitalis did not 
result in hemodynamic improvement, aldosterone 
secretion either remained unchanged or inci-eased 
in association with the deterioration in cardio- 
vascular function. 

Cardioglobulin and Related Studies 

The Physiological Effects of Cardioglobulin on 
Mammalian Hearts 

The nature of cardioglobulin, a protein system 
present in plasma of certain animals which exerts 
a cardiotonic effect on frog heart, has been dis- 
cussed in detail in previous reports. This year 
much of the effort has been directed at examining 
its effects on isolated mammalian cardiac tissue. 
In association with these studies the mechanism 
of its destruction in plasma as evidenced by a de- 
crease in the biologic activity of the prej)aration 
was also studied. 



110 



ANNTJAL REVIEW OF ESTTRAMURAL RESEARCH 



It has been established that right ventricular 
strips from guinea pig heart respond to cardio- 
globulin in a manner similar to that of frog heart. 
Eestoration of contractility and the ultimate de- 
velopment of contracture were noted in both 
species. Less dramatic effects were observed when 
papillary muscle of cat or right ventricular strips 
from rat were used. In these contracture did not 
occur although contractility of the hypodynaniic 
tissue was markedly improved. This is of interest 
since neither frog nor guinea pig plasma contains 
cardioglobulin. In contrast rat and cat plasma 
possess the system. The concentration of cardio- 
globulin used in the experimental studies in the 
latter animals was probably no greater than that 
normally present in their circulating blood thereby 
accounting for the less dramatic effects of the 
added substance. 

Cardioglobulin Degradation 

Studies on the degradation of cai'dioglobulin 
have centered on the observation that its activity 
on frog heart decreases rapidly under a variety 
of circumstances. Simple dilution in Ringer's 
solution at 37° C. is sufficient to eliminate cardio- 
globulin activity within 10 minutes. Of singular 
interest is the observation that destruction may be 
prevented entirely by addition of creatine phos- 
phate, though not by ATP, ADP, UTP, creatine, 
inorganic phosphate, or 10% albumin. Further- 
more, the inactive material may be reactivated 
by the addition of creatine phosphate at 37° for 
10 minutes. It has also been found that inactiva- 
tion of the cardioglobulin system is accomplished 
in undiluted plasma merely by the addition of a 
variety of tissue fractions, including crude tissue 
homogenates, pure myosin plus supernatant of 
tissue homogenates, or glycerinated muscle fibres. 
Under these circumstances inactivation is pre- 
vented not only by creatine phosphate but also by 
ATP or ADP. These provocative studies are 
being pursued actively since they may provide 
information concerning the chemical nature of the 
cardioglobulin system. 

Ryan/)dine-Digitalis Antagonism 

In the course of studies of isolated heart muscle 
it was observed that ryanodine, a water soluble 
plant alkaloid, was capable of depressing cardiac 
contractility in a manner similar to that effected by 



prolonged perfusion of the tissue with Ringer's 
solution. These studies were initially performed 
using rat right ventricular tissue. Unlike the hy- 
podynamic state produced by perfusion with 
Ringer's, which is reversed by the addition of 
cardiac glycosides, the ryanodine effect was unal- 
tered by the subsequent addition of digitalis-like 
compounds. Furthermore, the addition of ryano- 
dine alone to a digitalized ventricle abolislied the 
action of the glycoside, clearly demonstrating an 
antagonistic effect of these drugs. The diminu- 
tion in contractility provided by ryanodine can be 
reversed by perfusing the muscle with a solution 
deficient in either potassium or sodium. It is pre- 
sumed that this process reduces the cationic con- 
tent of the tissue. The results have been inter- 
preted in the framework of a hypothesis that an 
increase in cationic content of cardiac muscle 
is generally responsible for a diminution in 
contractility. 

Of particular interest was the observation that 
ryanodine not only antagonizes the digitalis-effect 
on contractility but also is capable of abolishing 
ventricular arrhythmias characteristic digitalis 
toxicity in both the intact cat and dog. Appro- 
priate concentrations of ryanodine were protective 1 
in animals to which otherwise lethal amounts of ■ 
digitalis had been administered. In contrast to 
the antagonism with respect to ventricular muscle 
arrhythmias, ryanodine and digitalis in combina- 
tion resulted in sinus node depression. The effect 
was abolished by atropine and it was concluded 
that the synergistic response to these derivatives 
on the sinus node was mediated by the vagus. 



LABORATORY OF CLINICAL BIOCHEMISTRY 

Amine Biogenesis and Metabolism 

It is in this area of amine formation and metab- 
olism that a rational approach to the development 
of pharmacologic agents is being attempted. For 
instance, from studies of tlie enzyme mechanism 
involved in the biosynthesis of norepinephrine it 
should be possible to devise inhibitors which by 
blocking one of the catal5'tic steps would produce 
a chemical sympathectomy. One of the enzj'mes 
involved in norepineiDhrine formation, aromatic 
L-amino acid decarboxylase, has now l)ecn well 



NATIONAL HEART INSTITUTE 



111 



characterized and potent inliibitors have been pre- 
pared which are effective in vivo, even in man. 
However, it is now extremely doubtful whether 
the endogenous production of norepinephrine can 
be limited in intact animals even by the most active 
decarboxylase inhibitor. If one presents the se- 



quence of reactions leading from L-tyrosine to nor- 
epinephrine in the form of a closed system flow 
diagram showing an estimated flow rate at each 
step, then it becomes apparent that the decarboxy- 
lase is far from being one of the rate-limiting 
steps in the reaction sequence. 



Tyrosine 



Tyrosine 



DOPA 



Dopamine 



Noradrenaline 



Hydroxylase 



Decarboxylation 



/3-oxidase 



Relative Activities of Catalytic Steps in Noradrenaline Biosynthesis 



Obviously even if decarboxylation of dopa is 
ioliibited over 90% the amoimt of dopamine, the 
end product of decarboxylation, would not become 
limiting. This explains why even the most potent 
of decarboxylase inhibitors, a-methyl dopa hy- 
drazine (MK 485 — Merck Sharp & Dolime) , does 
not lower tissue levels of norepinephrine or alter 
the excretion of the hormone or its metabolites. 
Other decarboxylase inhibitors, Aldomet, a-methyl 
dopa and a-methyl meta tyrosine, have been shown 
to lower tissue norepinephrine levels but by a 
mechanism which influences the binding sites. It 
does not appear worthwliile to devote more time 
to the development of pharmacologic agents based 
on decarboxylase inhibition. 

There are, however, two additional steps in the 
biosynthesis of norepinephrine, either of which 
can be made rate limiting. The ring hydroxyla- 
tion is not well characterized but the side-chain 
hydroxylation is now known to be catalyzed by 
an enzyme, dopamine-yS-oxidase, which appears to 
be distributed in sympathetically innervated tis- 
sues. Dopamine-/3-oxidase has been purified and 
its requirements elucidated (Kaufman et al.). A 
simple and specific assay has been developed in 
our laboratories based on the oxidation of tyra- 
mine to octopamine. Following this it was shown 
that the enzyme is not highly selective in its sub- 
strate requirements. Phenylethylamines of all 
types can be hydroxylated to yield the correspond- 
ing /3 substituted alcohols. Even mescaline can 
be converted to mescalol. All of these substrates 





H 


H 


R- 


-c- 


-c- 




H 


H 



can inliibit dopamine oxidation in a competitive 
mamier but since they have lower affinities would 
not be expected to be effective in vivo. It seemed 
that phenylethylamine isosteres with the follow- 
ing side chains would be more effective and per- 
haps irreversible inhibitors of the enzymes : 



-NH2 phenylethylamine (substrate) 



H 

E — C — N — NH2 benzylhydrazine (inliibitor) 
H H 

H 

E— C — O — NH2 benzyloxylamine (inhibitor) 
H 

H 
E-C-S-NH2 
H 

Several of these derivatives were therefore ob- 
tained and tested and found to inliibit the enzyme 
effectively at 10"'* M, the inhibition appearing to 
be of a non-competitive nature. It remains to be 
seen whether these are effective agents in vivo. 
One or two compounds related to those listed alx)ve 
were made available to the Laboratory of Chemi- 
cal Pharmacology by a drug company for other 
reasons, and they have obtained evidence which 
suggests that they can block norepinephrine for- 
mation in animals in vivo. MVe have shown that 



112 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



their compounds are potent inhibitors of purified 
dopamine-/8-oxidase. Thus it would appear that 
another type of pharmacologic agent may become 
available for blocking sympathetic activity. Dr. 
Albert Sjoerdsma is already preparing method- 
ology to carry such compounds into man should 
the animal biochemistry and toxicity data warrant 
it. 

The non-specificity of dopamine-/3-oxidase pre- 
sents another interesting problem. It has been 
shown that tyramine is normally present in tissues 
and can be oxidized to norsynephrine. Eecently 
we have administered tyramine and norsynephrine 
to animals and shown they are converted to nor- 
epinephrine and normetanephrine which are ex- 
creted in the urine. This is then an alternate 
route of norepinephrine biosynthesis. 



Tyrosine 

i. 

Tyramine 

I 



->-dopa 

I . 

-> dopamine 

I 



Norsynephrine >■ norepinephrine 

All of these steps have been demonstrated to 
take place in vivo and all the metabolites have 
been found in tissues. In fact, the only one whose 
presence in tissues is still questioned is dopa. The 
significance of this alternate route of norepi- 
nephrine biosynthesis is being investigated. 

Some of the same considerations which appear 
in norepinephrine formation also hold for sero- 
tonin biosynthesis. Although decarboxylase in- 
hibitors can block conversion of 5-hydroxytryp- 
tophan to serotonin the rate limiting step in vivo 
is the hydroxylation of tryptophan to 5-hydoxy- 
tryptophan. For this reason endogenous forma- 
tion of serotonin cannot be lowered by even potent 
decarboxylase inhibitors. As for tryptophan hy- 
droxylase, investigators at the University of Wis- 
consin recently demonstrated that liver homoge- 
nates could hydroxylate tryptophan. We were 
able to corroborate this but found that the enzyme 
responsible for this is actually L-phenylalanine hy- 
droxylase. Preparations of the phenylalanine en- 
zyme, purified according to Mitoma and to Kauf- 
man, were found to hydroxylate tryptophan. 
However, the affinity for tryptophan is about 0.01 
that of phenylalanine. In fact, the affinity is so 



low that it requires 10"= M solutions or greater of 
tryptophan to detect the activity. That one and 
the same liver enzyme hydroxylates phenylalanine 
and tryptophan is shown by : 

1) The same two enzymes responsible for 
phenylalanine hydroxylation are needed for 
tryptophan. 

2) The same pteridine cof actor is needed 
by both. 

3) Pteridine antagonists inhibit both. 

4) The ratio of phenylalanine to trypto- 
phan activity remains constant during purifi- 
cation. 

5) Competition can be shown between the 
two substrates, L-phenylalanine with the liigh- 
est affinity being a very effective inhibitor of 
L-tryptophan hydroxylation. 

These findings made it necessary to reinvesti- 
gate the localization of the hydroxylase in tissues. 
However, even with the most sensitive methods 
the hydroxylase could be demonstrated only in 
liver. If this enzyme were responsible for the 
serotonin found in brain, intestinal tract, lung, 
etc., it would mean that S-hydroxytiyptophan is 
transported to these organs via the blood from 
the liver. No evidence of circulating 5-hydroxj^- 
tryptophan can be obtained. Furthennore, 
studies in many laboratories indicate that sero- 
tonin-containing tissues can form serotonin from 
tryptophan (malignant carcinoid, etc.). We 
have administered methotrexate, a potent pteri- 
dine antagonist of liver phenylalanine hydroxy- 
lase, and were able to achieve marked inhibition 
of the liver enzyme with respect to both phenyl- 
alanine and tryptophan hydrozylation. Under 
these conditions tyrosine tissue levels fell signifi- 
cantly. However, no changes in serotonin tissue 
levels were observed. Tliese and other studies 
have convinced us that the liver hydroxylation of 
tryptophan is merely a non-specific activity which 
is observable with isolated purified enzyme prep- 
arations but which has little physiological signifi- 
cance. The tissue catalyst responsible for trypto- 
phan hydroxylation in serotonin containing ani- 
mal tissues must still be found. 

As for amine metabolism the enzyme, mono- 
amine oxidase (JIAO) is still under investigation. 
It has been shown that MAO is unique among 
deaminases because it can act on N-dimethyl sub- 
strates and is inhibited by iproniazid and related 



NATIONAL HEART INSTITUTE 



113 



compounds. In spite of these unusual properties 
MAO lias been shown to act in a manner similar 
to other deaminases, by dehydrogenation and addi- 
tion of water to an imino intermediate. The en- 
zyme has been purified to an extent where bmding 
of hydrazine inhibitors may help elucidate the 
active site of the enzyme. 

One final point of interest in the field of amines 
is the study of the vapor phase chromatography 
of amines. These procedures will make it pos- 
sible to study normally occurring amines, such as 
phenylethylamine, for wliich chemical assay pro- 
cedures are not available. Amine drugs such as 
amphetamine will also be made amenable to study. 

Homocamosine and Camosine Metabolism 

Since the finding of y-aminobutyrylhistidine 
(homocamosine) in brain the carnosines have been 
mider active study. Although a "camosine syn- 
thetase" has been reported this activity seems to 
be too weak (particularly in brain) to account 
for the large amounts of the camosine in tissues. 
Another possible mechanism for homocamosine 
formation would involve formation of y-glu- 
tamyl-histindine which, on decarboxylation, would 
yield homocamosine. Following this suggestion 
it has been possible to demonstrate a transpepti- 
dase enzyme in braia which can form y-glutamyl 
histidine from glutathione and histidine. It re- 
mains to be seen whether this peptide can undergo 
decarboxylation. 

During these investigations the need arose for 
measuring other peptides. Procedures for vapor 
phase chromatography of some di and tripeptide 
derivatives have been developed. 

Choline Biogenesis 

This problem which was dormant for about a 
year has been reactivated. In order to facilitate 
enzyme studies on cephalin formation it was neces- 
sary to elaborate new analytical procedures. It 
has now been possible to develop procedures for 
phosphatidyl serine and phosphatidyl ethanol- 
amine in tissue extracts which are specific and 
highly sensitive. Most important these proce- 
dures permit the assay to be made within a work- 
ing day whereas former methods for these com- 



pounds, besides being less specific, required several 
days. 



Collagen and Hydroxyproline 

Studies with intact chick embryos have shown 
conclusively that collagen formation occurs in the 
microsome fraction. Following administration 
of proline-C^* the microsomal "collagen" was 
foimd to contain much more hydroxyproline-C^* 
than any other collagen fi-action. Incorporation 
into the free hydroxyproline of the embryo was 
extremely low showing that it arose mainly as a 
decomposition product of the most slowly "tum- 
ing over" form of collagen. 

The studies were extended to cell-free systems 
and it has been shown that when chick embryo 
microsomes are incubated with proline- C^* in the 
presence of an ATP generating system, Mg** and 
the soluble cell fraction, hydroxyproline-C^*, ap- 
pear in the microsomes. Tliis hydroxyproline is 
present in a protein which is non-dialyzable and is 
extractable with hot tricliloroacetic acid as is col- 
lagen. Although the protein has not yet been 
more rigorously identified it would appear that it 
represents cell- free synthesis of collagen. It is of 
interest that even in this cell-free system proline 
is incorporated into collagen hydroxyproline far 
better than hydroxyproline itself. Preliminary 
studies indicate that it may be possible to disso- 
ciate the hydi'oxylation from the protein synthesis. 
It is hoped to increase the activity of the system 
and extend these studies to determine intermedi- 
ates and the nature of the catalysts. Studies with 
O^^ are nearing completion to determie whether 
the oxygen in hydroxyproline is derived from 
HaO^^ or from 02^^. Tliis will help in planning 
studies with the cell-free system. 

Actinomycin I, which is elaborated by S. anti- 
tiotious, is the only other hydroxyprolyine peptide 
which can be obtained m quantity. Studies with 
this organism indicate that proline-C^* is con- 
verted to peptide hydroxyproline (actinomycin I) 
and peptide ketoproline (actinomycin V) . It was 
found, however, that luilike the situation in col- 
lagen synthesis hydroxyproline can serve as a 
precursor for actinomycin I just as effectively as 
proline. Although this would indicate the pres- 
ence of a hydroxyproline activating enzyme such 



114 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



activity has not yet been demonstrated in cell-free 
preparations of the organisms. Attempts are be- 
ing made to demonstrate conversion of proline to 
hydroxyproline by cell-free preparations of 

/S. antibioticus. 

Amino Acid Transport 

Studies on the uptake of amino acids by brain 
have continued. It has been shown that the fea- 
tures which mark brain uptake as being under 
catalytic control are not foimd in peripheral tis- 
sues such as muscle. One of the consequences of 
this is that uptake of amino acids by brain is sub- 
ject to competitive inhibition to an extent which 
is not characteristic of most other tissues. Thus 
some of the central manifestations of phenylke- 
tonuria and "branched-chain" amino aciduria may 
be due to the high concentrations of circulating 
phenylalanine or leucines which inliibit uptake of 
related amino acids that are essential to brain 
function. 

Brain slices also differ from muscle slices in 
being able to concentrate a-amino acids. In addi- 
tion to the requirement for sources of energy it has 
now been shown that certain metabolic inhibitors 
and digitoxin inhibit this uptake. 

Biosynthesis of the B12 Coenzyme 

Conversion of vitamin B12 to its coenzyme form 
involves the substitution of an adenosyl moiety in 
place of the cyanide and reduction of the cobalt. 

ON 
\l/ 
CO+i 

/l\ 

Vitamin B12 
Adenosyl 

C0+> 
/l\ 
Coenzyme Bu 

It has now been shown that cell-free extracts of 
Clostridium tetanomoi'phum catalyze this con- 
version in the presence of ATP, glutathione and a 
yeast extract. Studies with labeled cyanide-C^' 
indicate that the ATP starts a concerted reaction 
involving reduction of the cobalt, release of cya- 
nide and attachment of the adenosyl moiety. The 
latter is derived from the ATP as is the ribose 
moiety. The enzyme system has now been purified 
many fold. 



CLINICAL ENDOCRINOLOGY BRANCH 

The work of the Clinical Endocrinology Branch 
has included chemical, physiologic and clinical 
studies related to the physiology of the adrenal 
cortex, biologic and clinical studies of calcium and 
phosphorus metabolism and of the physiology of 
the parathyroid glands, and several studies not 
closely related to these two areas. 

Studies of methodology in relation to adrenal 
physiology include the development of gas chro- 
matographic methods for the identification and 
measurement of aldosterone and otlier adrenal 
steroids, the biosynthetic production of aldoster- 
one ring-labeled with C" and its utilization for a 
double derivative isotopic method for analysis, 
studies in the degree of binding of aldosterone and 
other steroids to plasma proteins and to red cells 
and of various factors influencing this binding, 
and studies to imjDrove methodology for measure- 
ment of aldosterone secretion rates and of the in- 
fluence of various factors on these rates. 

By converting C21 steroids to acetate derivatives 
it was possible to measure and identify them by 
gas chromatography. Studies in recovery and 
degradation of these steroids were begun with the 
use of isotopically labeled precursors. Biosyn- 
thetically prepared C"-labeled aldosterone could 
be isolated in purified form, and pi-oved to be mod- 
erately stable. The substance was adequate for 
use in isolation procedures where a ring-labeled 
tracer was desirable to allow complete measure- 
ment of hydrolysis and recovery ; over the course 
of months the substance undergoes spontaneous 
degradation. 

Red blood cells and red blood cell ghosts were 
fomid to bind aldosterone and hydrocortisone, and 
the binding was readily reversible by washing 
with saline. Hydrocortisone was less bound to 
red cells than aldosterone as a result of a stronger 
affinity of plasma protein for hydrocortisone. In 
man, plasma protein is more effective in binding 
hydrocortisone than in the dog. Tliere is weak 
binding to albumin and very strong binding 
to corticosteroid-binding globulin. Aldosterone 
showed only the weak type of albumin binding. 
It was found that corticosterone. compound S, 
cortisone and prednisolone resembled hydrocorti- 
sone in their method of binding, wliereas pro- 
gesterone, desoxycorticosterone and 17 ketoster- 
oids resembled aldosterone in tlie absence of 



NATIONAL HEART INSTITUTE 



115 



specific binding. A method of titration of plasma 
with a given steroid was devised to measure and 
compare degree of binding as between steroids 
and as between clinical states. It was shown that 
whereas treatment with estrogen markedly in- 
creases the amount of corticosteroid-binding pro- 
tein (or the number of available sites), it is 
decreased in patients with hypoproteinemia. 

Secretion rates of aldosterone determined by 
measuring the decrease of specific activity of 
urinary steroid after intravenous administration 
of tracers confirmed in every respect conclusions 
derived from studies of urinary aldosterone ex- 
cretion. This method was used to study the effect 
of expansion of intravascular volume and the ef- 
fects of ACTH in extended studies. Preliminary 
results suggest that secretion rates determined 
over six hours may give information comparable 
to that acquired over twenty- four hours. 

Factors controlling adrenal steroid secretion 
were extensively studied. The role of the kidney 
was investigated, and the metabolic actions of 
renin and angiotensin were studied. In other 
studies the effect of changes in magnesium intake, 
of sustained treatment with ACTH, and of fast- 
ing were investigated and diurnal fluctuations of 
adrenal secretion were measured. Patients with 
primai-y aldosteronism were studied with a spe- 
cial reference to the effects of factors known to 
influence aldosterone secretion physiologically. A 
new syndrome, characterized by aldosteronism, 
hyperplasia of the juxtaglomerular apparatus and 
normal blood pressure was extensively studied in 
two patients, and a number of patients with the 
secondary aldosteronism of renal tubular acidosis 
were studied on metabolic regimen. 

Aldostei'one, corticosterone and Cortisol secre- 
tion, which are all lowered by hypophysectomy, 
fell to values sigTiificantly lower than those found 
with hypophysectomy alone when the kidneys 
were removed from dogs. When renal ischemia 
was produced by constriction of the renal arteries 
or of the lower aorta, secretion of aldosterone, 
corticosterone and Cortisol was increased. With 
moderate constriction it was possible to induce in- 
creases of corticosterone and Cortisol secretion 
without increases in aldosterone secretion. In 
animals in which aldosterone seci-etion had been 
increased by caval constriction, removal of the 
kidneys from the circulation lowered aldosterone, 

643351—62 9 



corticosterone and Cortisol secretion, and the resto- 
ration of the kidneys to the circidation restored 
the secretion to control values. These studies sug- 
gested that renal ischemia might have its effect 
through production of renin, and the effect of 
renin was accordingly measured. It was found 
that renin would induce increases in the secretion 
of aldosterone corticoserone and Cortisol in the 
hypophysectionized, nephrectomized dog. Quan- 
titatively the effects on the secretion of corticos- 
terone and Cortisol were relatively greater than 
those on the secretion of aldosterone. Synthetic 
asparagine 1 valine 5 angiotensin II was shown to 
reproduce the effects of renin, even at doses too low 
to induce changes in blood pressure: secretion of 
aldosterone, corticosterone and Cortisol was in- 
creased, but the relative increase of secretion of 
corticosterone and Cortisol was much greater than 
that of aldosterone. When angiotensin was given 
in small doses to normal human subjects over 
periods of 16 hours or more, they showed an 
increase in urinary aldosterone and moderate 
retention of sodium. At comparable doses, angio- 
tensin did not produce sodium retention in patients 
with Addison's disease. In large doses (sufficient 
to induce hypertension) angiotensin induced 
sodimn retention in normal subjects and in 
patients with Addison's disease. Direct measures 
of the effect of angiotensin on the isolated frog 
skin suggested that has no direct influence on 
sodium transport. It is likely that the sodium 
retention observed with angiotensin in patients 
with Addison's disease is secondary to the hemo- 
dynamic changes in the renal circulation. 

Changes in magnesium intake and total fasting 
had little effect on aldosterone excretion despite 
marked sodium loss with the latter procedure. 
Secretion of aldosterone, like the excretion of al- 
dosterone, was increased with ACTH but spon- 
taneously fell despite continuance of the ACTH. 
In some patients the secondary fall was associated 
with expansion of the intravascular volume, but 
in others the fall occurred without change in in- 
travascular volume. It is concluded that the 
secondary fall of aldosterone secretion does not 
require an increase in intravascular volume or in 
arterial pulse pressure. Excretion of aldosterone 
and of Porter-Silber steroids, as well as that of 
water and electrolytes were shown to undergo 
marked fluctuations during the day. For aldos- 



116 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



terone, Porter-Silber steroids, sodium and potas- 
sium, the maximum excretion occurred towards 
the middle of the day with minimal figures at 
night. The phase relationships were such that 
aldosterone might control the diurnal excretion of 
potassium, but was 12 hours out of phase to offer 
an explanation for diurnal changes in sodium 
excretion. 

Patients with primary aldosteronism were 
studied for the effects of restoration of body potas- 
sium, and of expansion of intravascular volume 
on aldosterone secretion and excretion. It was 
found that expansion of intravascular volume had 
little effect on aldosterone excretion or secretion. 
In contrast this procedure effectively decreased ex- 
cretion and secretion of aldosterone in patients 
with secondary aldosteronism. Two patients with 
primary aldosteronism and normal blood pressure 
were found to have marked hypertrophy and hy- 
perplasia of the juxtaglomerular apparatus. It 
was found that these patients manifested increased 
circulating levels of angiotensin and resistance to 
the hemodynamic action of antiotensin, a finding 
which suggests that this syndrome may result 
from a vascular insensitivity to angiotensin. Pa- 
tients with renal tubular acidosis were studied 
and found to have secondary aldosteronism, which 
was thought to be responsible for the potassium 
loss. Metabolic studies suggest that the primary 
defect in this condition is related to the inability 
of the renal tubule to produce a gradient in hydro- 
gen ion concentration between interstitial fluid 
and tubular lumen. There results a secondary 
inability to reabsorb Na with consequent aldos- 
teronism and increased secretion of potassium. 

Studies on calcium and phosphorus metabolism 
and on the physiology of the parathyroids 
included studies of the metabolic fate of vitamin 
D, studies of the effects on reabsorption of phos- 
phate of sustained phosphate loading and meas- 
urements of the role of the parathyroids in the 
release of bone calcium and the reabsorption of 
calcium by the renal tubules. 

Clinical studies included measurements of the 
defect in calcium metabolism in sarcoidosis, evalu- 
ation and development of tests for hyperparathy- 
roidism, and studies of the metabolic defects in 
renal osteitis and in idiopathic osteoporosis. 

Labeled vitamin D was obtained and studies 
were instituted to determine its biologic fate in 



bile fistula rats. Noraml and parathyroidecto- 
mized dogs were studied for phosphate reabsorp- 
tion and maximal tubular phosphate reabsorption 
during the administration of large loads of phos- 
phate orally over periods of several weeks. The 
reabosrption of phosphate decreased consistently 
in the normal dogs. A similar change was noted 
in the parathyroidectomized dogs but this was 
associated with marked clinical deterioration and 
with decreases in the glomerular filtration rate. 
Parathyroidectomy was shown to decrease the 
availability of bone calcium to restore serum cal- 
cium ion concentrations which had been acutely 
depressed with chelating agents. Whereas cal- 
cium feeding or vitamin D could restore serum 
calcium values to normal after parathyroidectomy, 
they did not restore the normal availability of 
bone calcium. It is concluded that the parathy- 
roids exert a measure of control over the physico- 
chemical release of calcium ion from bone. Pre- 
liminary results suggest that parathyroid hormone 
may increase renal tubular reabsorption of 
calcium. 

Studies in patients with sarcoidosis confirm the 
finding of very low fecal calcium with high uri- 
nary and occasionally high senim calcium, changes 
which suggest hyperabsorption of dietary calcium. 
This effect could be grossly exaggerated by ad- 
ministration of vitamin D in small doses and could 
be prevented by administration of prednisolone in 
large doses. Prednisolone, indeed, would reverse 
the effect even when given together with vitamin 
D. It was shown that the defect does not result 
from increased levels of vitamin D in the circula- 
tion, a finding which suggests that it represents a 
true state of hypersensitivity to the vitamin. 

Tests of phosphate clearance, of calcium absorp- 
tion and of the effect of calcium infusion were 
evaluated in normal subjects and in subjects with 
hyperparathyroidism. Of the available tests, 
only the increase of calcium excretion on a low 
phosphate intake and the failure of urinary phos- 
phate to fall with calcium infusion or to "re- 
bound" after calcium infusion proved reliable for 
the diagnosis of primary hyperparathyroidism. 
Patients with the osteitis of renal failure were 
shown to have a moderate or marked block in 
gastrointestinal absorption of calcium. It was 
found that vitamin D in large doses might cure 
the osteitis, and studies are in progress to deter- 



NATIONAL HEART INSTITUTE 



117 



mine whether it restores also the defect in calcium 
absorption. Patients with idiopathic osteoporosis 
were studied with especial reference to calcium 
and phosphorus balance under the influence of 
steroids and parathyroid hormone. It was found 
that strontium did not induce a positive calcium 
balance in three subjects. 

Investigations in other areas included studies 
of the pathogenesis of atherosclerosis, with spe- 
cial reference to permeability of vessel walls, 
studies on the interaction of catechol amines and 
corticosteroids on blood pressure, studies in the 
defect of free water excretion in Addison's dis- 
ease, experimental induction of a physiologic state 
similar to that seen with inappropriate secretion 
of antidiuretic hormone, measurements of the 
metabolic fate of albumin in patients with hy- 
poproteinemia and studies of the effects of hyper- 
calcemia and hypercalciuria and of amphotericin 
on renal function. 

Studies in atherosclerosis involved measure- 
ments of the rate of passage of plasma protein 
into the wall of the dog aorta. It was found that 
the rate of passage of labeled albumin, like that 
of cholesterol, was directly related to the arterial 
blood pressure. The essential findings could be 
reproduced in vitro, and it was thus possible to 
show that pulsation is not necessary to induce the 
acceleration of transfer brought on by hyperten- 
sion. It was shown furthermore that it was not 
dependent upon tension in the arterial wall, as 
there were no significant differences between the 
findings in small and those in large aortas. The 
finding suggested that passive filtration of large 
particles plays a part in the development of ath- 
erosclerosis. The effect of catechol amines in rais- 
ing the blood pressure was measured quantita- 
tively in subjects with Addison's disease. Each 
patient was studied with no treatment, with saline 
alone, with saline and desoxycorticosterone and 
with hydrocortisone. The findings do not suggest 
a dependence of the magnitude of response to 
catechols upon the presence of adrenal cortical 
steroids. The defect in water metabolism was 
studied in patients with Addison's disease under a 
similar protocol. In addition, some patients were 
subjected to expansion of intravascular volume 
with salt-poor human albimiin. Significant in- 
creases in free- water clearance could be produced 
with saline or with albumin, with little further 



effect added by addition of steroids. It thus ap- 
pears that contraction of intravascular volume is 
of relatively greater importance than steroid de- 
ficiency in producing the defect of water excretion 
in Addison's disease. 

Normal subjects on fixed sodium intake were 
subjected to stepwise reduction in plasma tonicity 
by progressive increment in water intake during 
the constant administration of pitressin tannate 
in oil. The syndrome of inappropriate secretion 
of antidiuretic hormone could be reproduced in 
all its essentials, and the findings did not support 
the notion that normal subjects differ from pa- 
tients with this syndrome in having an ability to 
"escape" from the effects of pitressin. The meta- 
bolic fate of human serum albumin given intra- 
venously was followed in patients with the hypo- 
proteinemia of gastrointestinal protein loss and 
in patients with analbuminemia. The former 
group was shown to degrade administered albu- 
min abnormally rapidly whereas the latter group 
degraded it significantly less rapidly than normal 
subjects. In the former group there was less nitro- 
gen excreted than the amount predictable on the 
assumption of total degradation of administered 
albumin with deamination of the amino acids 
therein, and it was concluded that albumin de- 
graded in the gastrointestinal tract is reformed 
into new tissue protein. In the latter group nitro- 
gen excretion actually decreased with albumin 
suggesting that the albumin provided a stimulus 
to the oxidation of fat. Hypercalciuria and hy- 
percalcemia were found to induce reversible de- 
creases in urinary concentrating ability, and oc- 
casionally to impair sodium reabsorption and hy- 
drogen ion secretion as well. Amphotericin was 
found to induce reversible decreases in glomerular 
filtration rate and in renal plasma flow. 



EXPERIMENTAL THERAPEUTICS 
BRANCH 

A biochemical approach to cardiovascular phar- 
macology and therapeutics has again been empha- 
sized in investigations of this laboratory. In 
addition, increasing use has been made of con- 
ventional physiologic techniques in studies on the 
actions of drugs in experimental animals and man. 
Various findings will be considered under the fol- 



118 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



lowing headings: 1) biosynthesis and metabolism 
of aromatic amines, 2) action and metabolism of 
drugs, 3) metabolism of hydroxyproline and col- 
lagen and 4) miscellaneous. 

Biosynthesis and Metabolism of Aromatic 
Amines 

Using our specific assay procedure for urinary 
histamine, several aspects of the metabolism of 
this amine in man were studied. Normal ex- 
cretion values were found to be 21-65 ^g/day for 
males and 15-90 /j,g/day for females. Oral ad- 
ministartion of L-histidine produced an increase in 
urinary histamine and high levels were found in a 
patient with gastric carcinoid tumor and in 
another with uticaria pigmentosa. Oral adminis- 
tration of nomycin or succinyl sulfathiazole pro- 
duced no change in urinary histamine indicating 
its origin from tissues rather tlian intestinal flora. 
It was shown that the specific histidine decarboxy- 
lase of mouse mast cell tumors could be inhibited 
by the hydrazino analogue of a-methyl-dopa 
(3iK485) ; preliminaiy studies are underway to 
detennine whether this drug M-ill lower urinary 
histamine in man. 

Tyramine has been found to be present in some 
mammalian tissues and in human urine. From 
in vitro studies it is known that this amine is an 
excellent substrate for monoamine oxidase and 
that it may undergo /3-hydroxylation to yield nor- 
synephrine. The metabolism of tyramine in the 
intact animal is conjectural. Accordingly the fate 
of tyramine administered intravenously to 5 
human subjects was studied. About 90 per cent 
of the intravenous dose was reco\'ered in the urine 
as p-hydroxyphenylacetic acid and only a few per 
cent as unchanged amine. A minor increase in 
p-hydroxymandelic acid was observed and sur- 
prisingly a significant increase in dopamine also. 
The latter finding requires further evaluation 
since a role for tyramine in the synthesis of norep- 
inephrine is suggested. 

A highly sensitive technique for the assay of 
monoamine oxidase (MAO) was developed, using 
tryptamine as substrate. An extensive survey of 
MAO activity in human tissues was performed. 
In general the results paralleled those found in 
animals. In contrast to the situation in most spe- 
cies, however, the human heart was found to be 



rich in MAO activity. Successful application of 
the method has been made to sjjecimens of human 
jejunal mucosa obtained by peroral biopsy. Hy- 
pertensive patients were found to have normal 
levels of MAO; marked decreases in jejuiuil 
MAO were demonstrable following introveuous 
or oral administration of MAO-inhibiting drugs. 
Diminished jejunal i\IAO was found in patients 
with thyrotoxicosis, in correlation with previously 
demonstrated elevations in urinary amines. These 
findings do not explain the enhanced sensitivity 
to catecholamines in thyrotoxicosis. 

Further attempts have been made to decrease 
serotonin synthesis in carcinoid patients by use 
of inhibitors of aromatic-L-amino acid decarboxy- 
lase. The use of a-methyl-dopa for this purpose 
was found to be limited in most patients by hypo- 
tensive effects. A marked chemical effect has now 
been achieved with its hydrazino analogue 
(MK485) in two patients. Further studies w 
depend on availability of the conipound. 



Action and Metabolism of Drugs 



ue ■ 



The effects of several sympathomimetic amines 
on plasma levels of free fatty acids (FFA) and 
blood glucose were studied in normal subjects. 
The following alterations in the basic phenylethyl- 
amine structure were found to be associated with 
maximal potency in mobilizing FFA's: hj'droxyl 
substitution in para position and on the y8-carbon 
and the presence of a primai-y or secondary amine. 
Effects on FFA and blood glucose were dissociable. 
No difference in FFA response in normotensives 
and hypertensives to infusion of norepinei)hrine 
could be shown, iu spite of obvious differences in 
vascular reactivity. 

Following the observation that intravenous in- 
jections of several different sympathoniimetic 
agents produced typical flushes in carcinoid pa- 
tients and that this effect could be blocked with 
phentolamine (Eegitine) , the latter compound was 
evaluated for its effects on spontaneous fluslies. 
Marked symptomatic imin'ovement has been noted 
in four jDatients with Eegitine in oral doses uj) 
to 100 mg every 4 to 6 hours. 

Effective inhibition of monoamine oxidase in 
human hypertensives is associated with an ortho- 
static lowering of blood pressure. Hemodynamic 
studies in nine patients receiving the MAO in- 



NATIONAL HEART INSTITUTE 



119 



hibitor, MO-911 (Pargyline, Abbott), point to 
drug-induced diminution of compensatory vaso- 
constriction in the erect position and during 
exercise, with resultant decrease in calculated pe- 
ripheral resistance. As a possible corollary in 
animals we have demonstrated blockade of trans- 
mission through sympathetic ganglia in intact cats 
following administration of several different 
MAO inhibitors. However, by assaying directly 
the MAO activity in ganglia it could be shown 
that onset, duration and degree of enzyme inhibi- 
tion do not parallel the ganglionic blocking effects 
of these drugs. It seems unlikely that the gangli- 
onic effects in animals explain the blood pressure 
effects in man. 

Therapeutic evaluation of Pargyline has now 
been extended to thirty hypotensive patients who 
have received the drug for periods up to 15 months. 
The agent has the advantage of combining useful 
mood-enhancing effects with potency and freedom 
from sympatholytic effects. In a few patients 
marked weight gain secondary to increased appe- 
tite, and interference with sexual functions in the 
male have made the drug unacceptable. In twelve 
patients in whom enzyme inhibition has been fol- 
lowed for up to six months by frequent measure- 
ments of urinary tryptamine, no appreciable loss 
of chemical effectiveness has been noted. The 
agent continues to be a promising antihypertensive 
drug in situations where lowering of blood pres- 
sure is not required urgently. 

Another area of therapeutic interest with MAO 
inhibitors, the alleviation of angina pectoris, is 
receiving increasing attention. Detailed studies 
in several patients indicate the increases of pulse 
rate and blood pi-essure occurring during tread- 
mill exercise may be markedly obtunded with 
impunity by careful administration of an MAO 
mhibitor. Diminished cardiac work resulting 
therefrom is associated with increased exercise 
tolerance. Principles derived from these studies 
may explain some of the discordant results I'e- 
ported in the literature. 

Following the demonstration that the hypoten- 
sive and decarboxylase inhibiting activity of 
a-methyl-dopa resided in the levo-rotatory form 
( Aldomet) , we have adopted this form for further 
study in patients. Our initial impressions of use- 
fulness and potency have been confirmed. Advan- 
tages include effectiveness against all degi-ees of 



hypertension, frequent lowering of recumbent as 
well as standing blood pressure, smoothness of 
effect and low incidence of tolerance. A possible 
limitation is the occurrence of febrile reactions in 
four of approximately one hundred patients 
studied. In each case the reaction develo^Ded with- 
in two weeks of initiating therapy. In two cases 
reversible abnormalities in liver function were ob- 
served after the onset of fever. 

From studies with other decarboxylase inhibi- 
tors in man, it now appears that the lowering of 
blood pressure with Aldomet is unrelated to en- 
zj'me inhibition. The hypotensive effect may be 
due to direct depletion of central or pei-ipheral 
stores of catecholamines, as suggested by studies 
in LCB. In experiments on tlie uptake of sero- 
tonin and norepinephrine by human platelets in 
vitro marked inhibition of uptake could be pro- 
duced with reserpine but little if any effect was 
observed with a-methyl-dopa or its major metabo- 
lite, a-methyl-doj^amine. Thus the amine deple- 
tion resulting from administration of a-methyl- 
dopa may be on a different basis from that 
prodxiced bj' reserpine. Xo clues to account for 
variations in blood pressure responses in different 
patients have been found in studies on metabolism 
of the drug. Addition of MK485 to treatment 
with Aldomet failed to alter the blood pressure 
i-esponse in several patients though formation 
of urinary a-methyl-dopamine was completely 
inhibited. 

Metabolism of Hydroxyproline and Collagen 

Utilizing a specific method developed in this 
laboratory for assay of hydroxyproline (HPr), it 
has been established that peptide-bound HPr in 
urine is a useful index of collagen degi'adation. 
However, both dietary and renal factors must be 
considered to obtain valid information. That 
HPr-peptides may be absorbed from the gastro- 
intestinal tract was shown by marked rises in 
plasma and urinary levels following ingestion of 
large amounts of gelatin by normal volunteere. 
This is the first demonstration of absorption of a 
peptide in man. Furthermoi'e, considerable day 
to day variation of urinary HPr has been observed 
in normal controls and patients unless the dietary 
intake of HPr was greatly restricted. Study of 
se\eral patients in severe renal failure has reveale-d 



120 



ANNXTAL REVIEW OF ESTTRAMURAL RESEARCH 



marked elevation of plasma peptide-HPr under 
this circumstance. 

Measurements of HPr excretion of a large group 
of normal volunteers have been made with these 
factors in mind. Each subject was required to in- 
gest a special diet extremely low in HPr. Pre- 
liminary findings show a progressive decrease in 
the amount of HPr excreted in successive decades 
of life, suggesting that urinary HPr may be an 
indication of the "metabolic age" of an indi- 
vidual's collagen. 

At least three different metabolic pools of HPr 
have been demonstrated in rats by measurements 
of the specific activity of HPr following adminis- 
tration of radioactive proline. Similar studies ap- 
pear feasible in man using highly active H^- 
labelled proline. Knowledge of turnover rates of 
HPr in man would be useful in setting up experi- 
ments designed to study the effects of drugs on 
collagen metabolism. We have recently set up an 
extensive screening program using weanling mice 
to test the effects of various drugs. HPr inter- 
mediates and analogues on the synthesis of total 
body protein and collagen. 

Miscellaneous 

A technique was developed for surgical denerva- 
tion of rat spleen. The catecholamine content of 
this organ was shown to be completely dependent 
in an intact innervation. 

A fibrinolysis assay model was devised using 
urokinase as an activator. Other constituents 
were also of human origin. Testing of a large 
number of sera from normal volunteers and pa- 
tients with various diseases revealed eight 
instances of elevated fibrinolysis inhibitor activity. 
Of these eight, two were from patients with essen- 
tial hyperlipemia and three from patients with 
thyrotoxicosis. 

CARDIOLOGY BRANCH 

1961 was the first full year of operation of the 
Cardiology Branch. The research efforts of this 
Branch were divided into two sections, Clinical 
Physiology and Clinical Biophysics, but the clin- 
ical efforts of both sections were combined. The 
research activities of tlie section of Clinical Physi- 
ology can be divided into four chief areas. 



Dynamics of Ventricular Contraction 

The basic principles of muscular contraction 
have heretofore been related to the human heart 
only in indirect fashion, primarily because the 
precise measurement of ventricular volumes, ven- 
tricular dimensions, the force of ventricular con- 
traction, ventricular contractility, and aortic blood 
velocity have not been possible in man. Efforts 
have been directed first to the development of 
methods to measure these important parameters 
and then to the utilization of these measurements 
in the study of the dynamics of ventricular 
contraction. 

During 1961 the applicability of Starling's law 
of the heart to unanesthetized hiunan subjects was 
studied by means of serial biplane-angiocardiog- 
raphy utilizing injections of contrast material into 
the left ventricle. The end-diastolic and end- 
systolic volumes of the left ventricle were deter- 
mined from the angiocardiagrams and stroke 
volumes calculated by subtracting the end-systolic 
from the end-diastolic volumes. In patients in 
whom beat-to-beat variations in left ventricular 
end-diastolic volume occurred, these were accom- 
panied by corresponding changes in stroke volume 
and arterial pulse pressure. However, in patients 
in whom the left ventricular end-diastolic volume 
remained almost constant, the stroke volume 
showed little or no change. From these observa- 
tions it was concluded that the ventricular stroke 
volume is a function of the end-diastolic volume 
and that therefore Starling's liw of the heart is 
applicable to man. 

The mechanical properties of human ventricles 
in situ were studied at the time of operation by 
sewing a specially designed strain gauge to the 
ventricle of 12 patients. This device permits the 
measurement of the resting (diastolic) and active 
(systolic) tension exerted by that segment of myo- 
cardium to which it is attached. Measurements 
of tension can be carried out with the muscle at 
varying lengths. The resting lengtli-tension 
curves of the human myocardium were hypei'- 
bolic and convex to the length axis. As the myo- 
cardial fibers were stretched from resting length to 
approximately 150% of resting length an increase 
in the developed tension and in the rate of develop- 
ment of tension were obsen'^ed. AVith greater 
stretching tliere was a decrease in these variables. 



NATIONAL HEART INSTITUTE 



121 



A method for the precise, continuous measure- 
ment of ventricular dimensions in closed-chest, 
intact human subjects has long been sought. 
Small silver clips were sutured to the surface of 
either the right or left ventricle of 17 patients 
undergoing cardiac surgery. During the post- 
operative period cineradiography was carried out 
and the distances between the clips were then 
measured on each individual frame of the film 
which had been obtained at a rate of 30 frames 
per second. During the Valsalva maneuver there 
was a marked decrease in right ventricular size, 
which averaged 12% of control values. With re- 
lease of the Valsalva and a deep inspiration, there 
was evidence of a "rebound". Cardioactive 
amines, such as isoproterenol and norepinephrine, 
given to patients during the course of this study 
usually resulted in a decrease in ventricular end- 
diastolic distances. Exercise also produced de- 
creases in end-diastolic and end-systolic dimen- 
sions. This cineradiographic technique thus 
permits, for the first time, accurate and repro- 
ducible measurements of ventricular dimensions 
in man. It is anticipated that this approach will 
prove to be a useful one for this detailed study of 
ventricular dynamics in unanesthetized intact 
patients. 

Since observations in experimental animals 
have indicated that the rate of ventricular pres- 
sure rise is a function of myocardial contractility, 
it was thought that determination of the rate of 
change of ventricular pressure (dp/dt) would per- 
mit study of myocardial contractility in intact 
man. In order to eliminate artifacts, ventricular 
pressures were recorded by means of a catheter 
with a high fidelity micronmanometer mounted at 
its tip. The first derivative of ventricular pres- 
sure was continuously computed by means of an 
electronic differentiating analogue computer. 
Normal values for the maximum dp/dt in both 
ventricles were determined and the effects of 
cardioactive amines, exercise, atropine-induced 
tachycardia, emotional stimuli, myocardial fail- 
ure, and of myocardial depressant drugs have 
been studied. This technique affords a convenient 
and accurate assessment of myocardial contrac- 
tility in intact human subjects. 

Studies on the determination and significance 
of the instantaneous pulsatile blood velocity in 
patients by the double-lumen catheter technique 



devised by Fry have been continued. The use of 
operational amplifiers (Donner Computer Units) 
has replaced passive electric analog circuits and 
tape recording techniques for recording prime data 
have been adopted. The use of tape recording 
units has made possible the recomputation of the 
instantaneous velocity and permitted more ac- 
curate assessment of the contributing factore for 
the terms involved in the hydrodynamic equations. 
Studies have now been done in 35 patients with 
meaningful data and useful correlations being 
available from the majority. 

Although it is generally agreed that Starling's 
law applies to the heart-lung preparations and 
open-chest anesthetized dogs, there is considerable 
disagreement about the relevance of the Frank- 
Starling mechanism to the intact organism. An 
electromagnetic flow meter was placed around the 
ascending aorta of dogs and changes in left ven- 
tricular dimensions and left ventricular diastolic 
and intrapleural pressures were measured simul- 
taneously. The chest was closed and the dogs 
permitted to recover from the anesthesia. Left 
ventricular stroke work, mean and peak values of 
aortic blood velocity and acceleration, and left 
ventricular power, all increased as left ventricular 
size and filling pressures were augmented. Dur- 
ing infusion of norepinephrine at any given filling 
pressure and ventricular size all of these indices 
of ventricular performance increased. These ex- 
periments support the view that Starling's law and 
the ventricular function curve concept are appli- 
cable to the heart of the closed-chest, unanesthe- 
tized dog with an intact autonomic nervous 
system. 

Adrenergic Nervous System and Myocardial 
Function 

The roles of the two enzymes, catechol-o-methyl 
transferase and monoamine oxidase, in the inac- 
tivation of norepinephrine liave not been fully 
elucidated. In preliminary studies on the isolated 
heart of the dog utilizing H^-norepinephrine, it 
was shown that the major metabolic product is 
normetanephrine, which demonstrates the impor- 
tance of catechol-o-methyl transferase as an 
inactivating mechanism in cardiac tissue. 

Recently, on the basis of pharmacologic observa- 
tions it has been suggested that tlie action of some 



122 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



sympathomimetic amines is mediated by release of 
endogenous norepinephrine from adrenergic 
nerves. Since there has been little direct evidence 
to support this hypothesis, a study was under- 
taken to determine whether such vasoactive amines 
release norepinephrine from the heart. Tyra- 
mine, phenylethylamine, tryptamine, p-hydrosy- 
amphetamine, amplietamine, ephedrine, mephen- 
termine, and guanethidine, on intravenous 
administration, all produced release of norepine- 
phrine into the coronary venous blood. Phenyle- 
phrine, methoxamine, and serotonin, however, had 
no such effect. After infusion of tyramine for 
one hour the norepinephrine content of the heart 
was consistently reduced. These observations 
lend direct proof for the hypothesis that a number 
of vasoactive amines affect the cardiovascular 
system by means of release of endogenous 
norepinephrine. 

It has been established previously by investiga- 
tors in the Laboratory of Cardiovascular Physi- 
ology, NHI, that stimulation of the stellate gang- 
lion results in an efflux of norepinephrine into 
coronary sinus blood. In studies carried out in 
this laboratory, it was demonstrated that the myo- 
cardial norepinephrine content is not altered at a 
time when the physiologic effects of supramaximal 
cariod-accelerator nerve stimulation are markedly 
reduced, although at this time the postganglionic 
action potentials are unaltered. Hence, despite a 
normal tissue catecholamine store and delivery of 
the stimulus to the adrenergic neuro-effector junc- 
tion, the response of the effector organ is dimin- 
ished. These data provided indirect evidence for 
a high rate of local amine synthesis and/or myo- 
cardial extraction of circulating plasma catechola- 
mines. 

The acute effects of guanethidine and bretylium 
on heart rate, myocardial contractile force, arte- 
rial pressure and cardiac output have been studied 
in normal dogs and dogs with denervated hearts 
in which the myocardial catecholamines have been 
depleted. The strikingly positive inotropic, 
chronotropic, and pressor effects as well as the in- 
crease in cardiac output produced by guanethidine 
in normal dogs were markedly reduced in the dogs 
with denervated hearts. Denervation of the heart 
reduced the positive chronotropic effects as well 
as part of the increase in cardiac output produced 
by bretylium in the normal dogs. These data in- 



dicate that a large part of the acute heniod3'namic 
effects of guanethidine and bretylium are produced 
as a result of the sudden release of myocardial 
catecholamines. 

Although it has been generally accepted that 
the hemodynamic effects of tyramine are produced 
by sudden catecholamine release, the source of 
the catecholamine which produces the effects on 
the heart has not been determined. Experiments 
carried out on normal and reserpinized dogs and 
dogs with denervated hearts indicate that the re- 
lease of myocardial catecholamine stores is pri- 
marily responsible for the cardiac eff'ects of tyra- 
mine. Furthermore, these studies with tyramine, 
guanethidine and bretylium demonstrate the 
physiological potential of stored myocardial 
amines. 

Expei-iments were carried out in order to deter- 
mine how the mj'ocardial norepinephrine content 
modifies the effect of a digitalis glycoside on the 
heart. In experiments in which the myocardial 
responses to ouabain in normal and reserpinized 
dogs were compared, no differences either in the 
inotropic or the arrhythmogenic properties of the 
glycoside were observed in these two groups of ■ 
animals. However, it was noted that the func- | 
tional refractory period of the A-V node was 
markedly greater in the reserpinized llian in the 
normal animals and that although ouabain was 
capable of prolonging the refractory period in I 
normal dogs it failed to do so in reserpinized ani- 
mals. The infusion of norepinephrine into reser- 
pinized animals shortened the refractory period I 
of the A-V node, suggesting that tlie heart of the 
reserpine pretreated animal can bind circulating 
norepinephrine, and that this bound store is po- 
tentially active physiologically. 

In spite of the widespread clinical use of anti- 
adrenergic drugs, such as reserpine and giianethi- 
dine, little is known of the liemodynamic mecha- 
nisms undei'lying their hypotensive effects. The 
possibility was considered that one of the impor- 
tant mechanisms of their action was through their 
effect on blocking venous venoconstriction. 
Utilizing the major vessel occlusion technique for 
measuring reflex venoconstriction, it was found 
that both acute and chronic administration of 
guanethidine and of resej-pine blocked this reflex. 
Norepinephrine infusion failed to restore tlie re- 
flex venoconstrictor response. These observations 



NATIONAL HEART INSTITUTE 



123 



suggest that the action of these drugs on the ve- 
nous bed may play a prominent role in their clini- 
cal effects. 

In order to assess the contribution of the auto- 
nomic nervous system to the hemodynamic re- 
sponse to exercise, normal subjects were studied 
at rest and during steady-state leg exercise in a 
control period without drugs, and after pharma- 
cologic interference with the autonomic nervous 
system. The latter was accomplished by the ad- 
ministration of guanethidine and atropine. Fol- 
lowing the administration of these drugs, muscular 
exercise resulted in significantly smaller increases 
in heart rate, stroke volume, cardiac output and 
left ventricular work than during the control pe- 
riod. However, the arterio-mixed venous oxygen 
differences were substantially greater following 
autonomic blockade than in the control state. 
These data indicate that combined pharmacologic 
inhibition of the sympathetic and parasympathetic 
nen- ous systems interferes with the normal cardio- 
vascular response to exercise. 

Attempts have also been made to evaluate the 
contribution of adrenergic reflexes and myocardial 
catecholamine stores to cardiac homeostasis in man 
by measiiring the effects of guanethidine adminis- 
tration upon patients in borderline congestive 
heart failure. In 4 such patients guanethidine 
produced sodium retention and weight gain and 
three of these showed concomitant increases in 
venous pressure, hepatic enlargement, dyspnea, 
orthopnea and peripheral edema. These effects 
disappeared after discontinuation of guanethidine 
ti'eatment, and were produced by doses of guan- 
ethidine which did not cause significant hypo- 
tension. These results indicate that guanethidine 
administration can aggravate congestive heart 
failure in patients with overt decompensation and 
that interference with the adrenergic component 
of the autonomic nervous system is deleterious to 
patients with reduced cardiac reserve. 

Previous studies in this laboratory have sho-^vn 
that guanethidine reduces the tremor, restlessness 
and tachycardia associated with tri-iodothyronine 
induced hyperthyroidism in nonnal control sub- 
jects. These results were interpreted as evidence 
that the adrenergic reflexes are important in many 
of the manifestations of hyperthyroidism. 
These observations have been extended to include 
measurement of cardiac outputs in clinical hyper- 

643351—62—10 



thyroidism and in normal subjects with tri-iodo- 
thyronine induced hyperthyroidism. In 8 sub- 
jects with high cardiac outputs associated with 
endogenous or exogenous thyroid excess, guanetlii- 
dine lowered the outputs markedly in only two. 
This suggests that the high cardiac outjDut in 
hyperthyroidism is not dependent upon sympa- 
thetic reflexes. 



Cardiovascular Diagnostic Techniques 

Transseptal left heart catheterization, a tech- 
nique previously developed in the Heart Institute, 
was modified considerably during 1961. The pro- 
cedure is now performed by percutaneous punc- 
ture of the femoral vein. Left atrial puncture is 
carried out with a needle having a 21 gauge tip, 
thus decreasing the hazard of accidental puncture 
of a structure other than the atrial septum. A 
radiopaque catheter is then passed over the needle 
into the left atrium and across the mitral valve 
into the left ventricle. The lumen of the catheter 
is large enough to permit rapid injection of a large 
quantity of radiopaque dye and the accm'ate meas- 
urement of pressures in the left atrium and left 
ventricle. The numerous technical advantages of 
this modified technique have led to the almost 
universal acceptance of the transseptal method as 
the technique of choice. Investigators from nu- 
mei-ous laboratories learned the technique on visits 
to Bethesda. More than 500 such procedures have 
been performed here. Transseptal left heart 
catheterization in infants and children with heart 
disease, in children and adults without cardio- 
vascular disease and transseptal left heart angio- 
cardiogi-aphy received particular attention this 
year. 

In spite of the widespread applications of left 
heart catheterization, the pressures in the left side 
of the heart in subjects without cardiovascular dis- 
ease and in basal physiologic states have not been 
known. Transseptal left heart catheterizations 
were carried out on 18 individuals without organic 
heart disease. The mean left atrial pressures 
ranged between 2 and 12 mm Hg, with an average 
value of 7.9 mm Hg. The mean left atrial pres- 
sure exceeded the mean right atrial pressure by an 
average of 3.9 mm Hg. The left atrial Y peak 
averaged 12.8 mm Hg and the left ventricular end- 
diastolic pressure ranged froui 5 to 12 mm Hg 



124 



ANNTJAL REVIEW OF INTRAMURAL RESEARCH 



with an average value of 8.7 mm Hg. It is antici- 
pated that these values will serve as normal stand- 
ards for these parameters in future studies of left 
heart dynamics. 

It was shown previously that the inhalation of 
an inert foreign gas and the determination of its 
concentration in blood sampled simultaneously 
from the right side of the heart and the systemic 
arterial bed form the basis of a sensitive test for 
the characterization of left-to-right circulatory 
shunts. Radioactive krypton (Kr^^) has been 
found to provide substantial advantages over other 
gases and it has therefore been employed routinely 
in this laboratory for the past several years. In 
1961 the results of inhaled Kr^^ tests, carried out 
in 323 patients in whom the diagnosis was firmly 
established, were analyzed. In the 161 patients 
subsequently proved to have left-to-right shunts 
the results of the test ranged from 13% to 113%. 
In the 162 patients without cardiac shunts the re- 
sults ranged from 0.9% to 12.2%. The Kr^^ test 
may thus be employed with confidence for deter- 
mining the presence or absence of a left-to-right 
shunt. In addition, when the test is successively 
performed in the pulmonary artery, right ventri- 
cle, and right atrium, the site of entry into the 
right side of the heart may be correctly localized. 

A simple technique for the recording of indi- 
cator-dilution curves from the pulmonary vascular 
bed and its applications to the detection of left- 
tor-right circulatory shunts was developed. Dio- 
drast labeled with I^^^, a gamma-emitting isotope, 
was injected intravenously and its activity in the 
pulmonary vascular bed determined with a scintil- 
lation detector placed over the upper lung field. 
In the patients in whom the presence of a left-to- 
right shunt was subsequently proved, the descend- 
ing limb was prolonged. Simple analysis of the 
curves in a total of 33 patients allowed determina- 
tion of the presence or absence of a shunt. Placing 
the detector over the lung fields, rather than the 
precordium, markedly simplified the analysis of 
the curves and resulted in increased sensitivity. 
The clinical value of this simple technique in the 
study of patients following cardiac operations and 
in the screening of patients with heart murmurs 
of uncertain etiology was demonstrated. This 
technique is now used routinely for clinical studies 
at the NHI and at a number of other institutions. 



A technique has been developed for determin- 
ing the fraction of left ventricular end-diastolic 
volume which is ejected during each cardiac cycle. 
Radioiodinated Diodrast was rapidly injected into 
the left ventricle at the time of transseptal left 
heart catheterization and the fraction of isotope 
which was discharged from this chamber per beat 
was determined with a well-shielded scintillation 
probe placed on the chest wall over the left ven- 
tricle. Left ventricular end-diastolic volume was 
estimated from the stroke volume determined by 
the dye-dilution method, and the fraction of iso- 
tope discharged per beat. Difficulties resulting 
from inadequate mixing of isotope in the left ven- 
tricle were minimized by this technique since the 
probe detected indicator in the entire left ventric- 
ular cavity. The accuracy of this technique was 
first demonstrated in a circulatory model and then 
in open-chest dogs in which ventricular dimen- 
sions were continuously monitored by means of 
mercury-in-rubber gauges. In 21 patients with- 
out detectable abnormalities of left ventricular 
functions the fraction of left ventricular volume 
discharged averaged 37 ±8% per beat and the end- 
diastolic volumes averaged 89±26 ml/M^ B.S.A. 
In 21 patients with heart failure and/or valvular 
i-egurgitation, in whom left ventricular function 
was compromised, the fraction of left ventricular 
volume discharged into the aorta averaged 16 ± 
5% per beat and the end-diastolic volumes aver- 
aged 209 ±75 ml/M= B.S.A. This technique has 
been found to be sensitive to changes in left ventric- 
ular function and practical to apply routinely 
in the course of left heart catheterization. 

The most acurate technique available for the 
measurement of blood oxygen content is the mano- 
metric technique of Van Slyke. Unfortunately 
this method is a tedious one, and can be carried 
out only by a trained technician. In collaboration 
with the Laboratory of Technical Development, 
NHI, the rapid analysis of blood oxygen content 
by a gas chromatographic technique is being ex- 
plored. A rugged and very sensitive ionization 
detector designed to detect minute (less than 0.1 
microliter) quantities of O2 and other gases has 
been developed. This detector lias been sliown to 
have excellent accuracy and reproducibility over 
a relatively wide range. The extraction of oxygen 
from very small samples of blood has proved to 
be the major problem which is incompletely re- 



NATIONAL HEART INSTITUTE 



125 



solved thus far. The perfection of this technique 
would be of considerable importance to cardiac 
catheterization laboratories. 

The accurate measurement of systolic arterial 
pressure in infants and young children may be a 
formidable task. A modification of the Wliitney 
mercury strain gauge with an Eisner impedance 
matching circuit and a recording galvanometer 
has been developed into a simple and sensitive de- 
vice with which to record systolic arterial blood 
pressure in patients of all age groups. This tech- 
nique has been shown to be accurate to within 5 
mm Hg of the directly recorded systolic arterial 
pressure. The simultaneous recording of systolic 
blood pressures from the upper and lower extrem- 
ities by the use of two plethysmographs has proven 
useful in the pre- and postoperative evaluation of 
patients with coarctation of the aorta. This sim- 
ple but accurate device can be constructed for ap- 
proximately $40. 

The presence of an atrial (fourth) heart sound 
has been observed frequently in patients with se- 
vere aortic stenosis. This observation led to a 
careful phonocardiographic-hemodynamic corre- 
lation of this soimd. In a study of 50 patients 
with aortic stenosis it was found that the absence 
of this sound signifies that the aortic valve ob- 
struction is relatively mild, with a gradient less 
than 70 mm Hg. This sound, which may be de- 
tected clinically or recorded phonocardiographic- 
ally, thus appears to be very helpful in the assess- 
ment of patients with aortic stenosis. 

The time interval between the two components 
of the second heart sound and the changes in this 
interval during respiration constitutes one of the 
most important physical signs in the bedside di- 
agnosis of congenital heart disease. The mecha- 
nisms responsible for the behavior of this sound 
are still under debate. The characteristics of the 
second heart sound were analyzed in phonocardio- 
grams recorded from 350 patients in all of whom 
the diagnosis was proved either at operation or 
by detailed catheterization studies. Normal 
values for the time intervals between aortic and 
pulmonic valve closure, for the duration of right 
and left ventricular systole and for the effects of 
respiration and of the Valsalva maneuver on these 
intervals in patients without heart disease and 
with a variety of congenital lesions were estab- 
lished. These studies showed that careful auscul- 



tatory and/or phonocardiographic analyses of the 
effect of respiration on the two components of the 
second heart sound may help considerably in the 
diagnosis of all of the major forms of congenital 
heart disease. 

Clinical Cardiology 

During 1961 a number of clinical studies on 
patients with congenital heart disease were con- 
ducted in conjunction with the Clinic of Surgery, 
XHI. 

The clinical and hemodynamic features of a 
previously unrecognized variant of partial A-V 
canal were described. Two patients with evidence 
of massive mitral regurgitation due to cleft mitral 
valves were discovered to have small associated 
atrial septal defects of the ostium primum variety. 
The interaction of these two lesions resulted in a 
decompression of the distended left atrium by the 
defect, and the anomalous hemodynamic effects of 
the repair of these defects were described. 

The clinical, hemodynamic and angiocardio- 
graphic findings in two other unusual congenital 
cardiovascular anomalies were described in detail. 
Seven patients with isolated congenital mitral re- 
gurgitation and six patients with aortopulmonary 
septal defects were studied. Comprehensive 
studies on these patients permitted definitive de- 
scription of these two lesions, about which rela- 
tively little clinical or hemodynamic information 
was heretofore available. The decline in the in- 
creased pulmonary vascular resistance following 
surgical closure of the aortopulmonaiy septal de- 
fects in four patients were particularly interesting. 

Five patients with tetralogy of Fallot who de- 
veloped pulmonary hypertension following crea- 
tion of an aortopulmonary anastomosis were de- 
scribed in collaboration with members of the 
Department of Medicine, Johns Hopkins Medical 
School. Thus, for the first time the develoiDment 
of an elevated pulmonary vascular resistance in 
man could be related directly to increased pul- 
monary blood flow. The characteristic radiologic 
features in the five patients should permit ready 
recognition of this complication of sugical treat- 
ment for tetralogy of Fallot. 

A total of 27 patients with idiopathic hyper- 
trophic subaortic stenosis have now been studied. 
A number of new variants liave been recognized. 



126 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 






These include: 1) patients in whom the left in- 
traventricular pressure gradient is pi'esent on some 
occasions and absent at other times; 2) familial 
forms of the disease in Avhich some members of 
the family have obstruction to left ventricular out- 
flow, others obstruction to right ventricular out- 
flow and others with obvious left ventricular 
enlargement but without hemodynamic evidence 
of obstruction. The possibility was suggested 
that the same basic disease process may, in dif- 
ferent patients, be responsible for a variety of 
clinical and hemodynamic pictures. 

It is now clear that electrocardiographic evi- 
dence of a disturbance in right A^entricular con- 
duction with QKS prolongation develops in the 
majority of patients in whom an isolated ventricu- 
lar septal defect is repaired or in whom the 
teralogy of Fallot is completely corrected. The 
time intervals between the onset of ventricular de- 
polarization and of right ventricular contraction 
wei'e determined both before and after operation 
in 17 patients who developed electrocardiographic 
evidence of right ventricular conduction disturb- 
ance. It was concluded that the mechanical delay 
in the onset of riglit ventricular contraction which 
develops in the majority of patients following 
closure of ventricular septal defects and complete 
correction of tetralogy of Fallot results from in- 
terruption or of other trauma to the right bundle 
branch. 



Circulatory Physiology 

Myocardial oxygen consumption is being studied 
in the dog utilizing a preparation in which the 
external work of the myocardium is maintained 
near zero while coronary blood flow and oxygen 
delivery are varied. The heart is hemodynami- 
cally isolated from the systemic cix-cuit and the 
root of the aorta is perfused by a separate pump. 
The entire coronary venous return to the right side 
of the heart is diverted to a separate reservoir and 
coronary flow can be accurately determined. 
Myocardial oxygen consumption is determined 
from the product of coronary blood flow and the 
coronary ai'teriovenous oxygen dift'erence. In 1)>, 
experiments it has been demonstrated that as cor- 
onary blood flow is increased from low levels there 
is a progressive increase in myocardial oxygen 
consumption despite maintenance of constant ex- 



ternal left ventricular work. These observations 
demonstrate that myocardial oxygen consumption | 
may vary with varying coronary blood flow (and ' 
varying oxygen delivery) while the external work 
of the heart is kept constantly near zero and sug- 
gest that previously described determinants of 
myocardial oxygen consumption may be incom- 
plete because this phenomenon had not been 
appreciated. 

The effects of hypoxia on the capacity of the 
total systemic vascular bed, venous return, sys- 
temic vascular resistance, and myocardial contrac- 
tile force were studied in dogs. The efl'ects of 
hypoxia on each of these aspects of the cardio- 
vascular system was evaluated separately. In 
addition the role of the chemoreceptoi's in the 
mediation of these responses was investigated by 
hemodynamically isolating or denervating these 
structures. Generalized systemic hypoxia resulted 
in striking venoconstriction. Since this eifect 
could be prevented bj' chemoreceptor denei-vation 
it was concluded that it was mediated by tiie 
chemoreceptor reflex arc. An increase in systemic 
vascular resistance occurred when hypoxemia was 
localized to the carotid arterial bed. However, a 
decline in systemic vascular resistaiice was ob- 
serve when the entire systemic vascular bed was 
made hypoxic and the chemoreceptors denervated 
or perfused with oxygenated blood. These results 
indicate that the direct eifect of hypoxia is to pro- 
duce arteriolar dilatation but that this response 
is opposed by the arteriolar constriction which is 
mediated by the chemoreceptor reflex arc. Myo- 
cardial contractile force increased when intact 
chemoreceptors were perfused with hypoxic blood, 
regardless of whether the heart received well or 
poorly oxygenated blood. However, myocardial 
contractile force decreased whenever poorly oxy- 
genated blood perfused the heart and the chemo- 
receptors were either denervated or perfused with 
well-oxygenated blood. It therefore appears that 
the augmentation of myocardial contractile force 
which occurs during general izetl hypoxia is de- 
pendent upon an intact chemoreceptor reflex arc 
which opposes tlie direct myocai-dial depressant 
effects of hypoxia. 

Hypothermia has been produced in dogs on 
complete cardiopulmonary bypass at a constant 
systemic perfusion rate. Cooling to 15-25° C. lias 
been eft'ected. During tlie cooling phase a de- 



NATIONAL HEART INSTITUTE 



127 



crease in peripheral resistance (28%) , a decrease in 
venous return, and an augmentation of systemic 
blood volume (24 ml/kg body weight) have been 
noted. In addition to direct measurement of the 
systemic blood volume changes, an indirect dye 
dilution technique was utilized. A decrease in 
calculated circulating blood volume during hypo- 
thermia was demonstrated with the latter tech- 
nique. These observations indicate that during 
hypothermia, significant arteriolar dilatation oc- 
curred and that substantial volumes of blood were 
trapped in the peripheral vascular bed. The lat- 
ter resulted in the striking decreased in venous 
return and an increase in systemic blood volume. 
The acute hemodynamic effects of ouabain were 
studied in patients with valvular aortic stenosis 
and in patients with idiopathic hypertrophic sub- 
aortic stenosis. Left atrial and left ventricular 
pressures and cardiac output were measured. In 
the patients with valvular aortic stenosis, ouabain 
either improved left ventricular function or had 
no discernible effect on it, but in no patient was 
left ventricular function depressed. In the pa- 
tients with hypertrophic subaortic stenosis, the 
left ventricular end-diastolic pressure and mean 
left atrial pressure rose significantly following 
ouabain adminstration ; cardiac output either fell 
or remained unchanged and the systolic pressure 
gradient between the left ventricle and the brachial 
artery rose. These actions of ouabain in hyper- 
trophic subaortic stenosis are considered to result 
from a sustained increase in left ventricular con- 
tractile force which increased the obstruction pro- 
duced by the muscular outflow tract. 

Clinical Physiology 

A substantial fraction of the professional and 
teclinical efforts of the Section of Clinical Physi- 
ology of the Cardiology Branch is devoted to 
clinical activities which are luirelated to its re- 
search program. These non-research activities 
include: 1) Recording, mounting and interpreta- 
tion of all of the electrocardiograms for the Clin- 
ical Center. Approximately 6,000 tracings were 
handled in 1961. In addition a course in electro- 
cardiagraphic interpretation was given and per- 
sonal instruction in ECG interpretation was 
provided to Clinical Associates from other labora- 
tories of NHI and from other Institutes. 2) Clin- 



ical cardiology consultations for the Clinical 
Center. 3) Cardiology consultations to the Clinic 
of Surgery, NHI. 4) An average of 3 postopera- 
tive cardiac catheterizations weekly, carried out 
for the Clinic of Surgery. 5) Consultations in 
pulmonai-y phj'siology, and performance of pul- 
monary function tests for the Clinical Center. 

Clinical Biophysics 

A major activity of the Section of Clinical Bio- 
physics has been the development of realistic 
mathematical models of the circulatory and pul- 
monaiy systems. To the extent that this can be 
accomplished new hypotheses can be tested ex- 
perimentally and computer techniques can be 
brought to bear on solutions of many physiologic 
problems in health and disease. Progress in this 
area depends on an active program in instru- 
mentation development as well as comprehensive 
studies of the physical properties, anatomy, 
stresses (including pressures), tissue reactions to 
these stresses, and general system behavior (in- 
cluding feedback phenomena) of the circulatoiy 
and pulmonary systems. Therefore, the activities 
of this section may be summarized under three gen- 
eral headings: 1) instrumentation, 2) circulatory 
studies, 3) pulmonary studies. 

Ins t-)mt?}ientat ion 

The measurement of stresses in general and pres- 
sure in particular have continued to constitute a 
major effort of this section. A set of standards 
for the accurate measurement of vascular pressure 
and the axial blood vessel pressure gradient have 
been established. Results show that cui'rently 
available pressure transducers can be made to meet 
minimum standards for both of these measure- 
ments. Moreover the estimation of the pressure 
gradient is essentially independent of pi-essure tap 
separation up to values of 5 cm. 

The performance of the Kolin electromagnetic 
flowmeter and the computed pressure gradient 
technique were compared to a monitored pulsating 
flow. Both the Kolin electromagnetic flowmeter 
and the pressure gradient technique were sliown to 
have excellent recording fidelity for pulsatile flow 
up to at least 10 cycles per second. The pressure 
gi-adient technique was originally developed in this 
laboratory for the estimation of instantaneous flow 



128 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



in man since the necessary pressures are attainable 
by conventional catheter techniques. Preliminary 
results from studies in conjunction with investiga- 
tors working at the Mt. Alto Hospital indicate that 
the method gives reasonable values for blood flow 
and distribution of blood flow in man. 

Considerable effort was made to apjDly analog 
computer techniques to various systems of equa- 
tions describing particular biological systems. It 
it concluded that although analog computer tech- 
niques are fruitful for relatively simple systems, 
their lack of flexibility seriously limits the appli- 
cation of analog techniques to the more common 
complicated systems represented by the lung and 
circulation. Therefore, greater effort must be 
made to facilitate the use of digital computer 
techniques. 

Circulatory Studies 

Statements describing the circulatory system 
can begin with a description of the hydraulic load 
on the ventricles. An essential part of this descrip- 
tion is the hydraulic input impedance of the vascu- 
lar bed. Although studies in this area continue, 
results to date indicate that the systemic circula- 
tion is a distributed system, behaving in many 
ways like an electrical transmission line. The 
spectrum of input impedance to the aorta shows an 
amplitude that waxes and wanes with frequency 
and an associated phase angle that oscillates be- 
tween minus 90 degrees and plus 90 degrees. 

Similar studies in the pulmonary artery reveal 
that this system also behaves like a distributed 
system, in spite of its relatively short length. The 
explanation lies in the observation that the pulse 
wave velocity in the pulmonary artery is much 
lower than that in the aorta. Interpretation of 
hydraulic impedances in the pulmonary artery are 
complicated by the presences of nonlinear terms 
in the equations of fluid motion. The significance 
of these terms, which are the result of the highly 
nonuniform geometry of the pulmonary fluid 
boundaries, has been evaluated. 

Concomitant with the foregoing studies of the 
cardiac load, attempts have been made to define 
the manner in which myocardial contraction will 
deal with this load. The force-velocity equation 
of A. V. Hill was transformed into a pressure-flow 
equation applicable to the heart. On the basis of 
preliminary experiments, it appears that there 



exists a miique instantaneous pressure and flow for 
a given instantaneous fiber length. 

Pulmonary Studies 

Theoretical considerations have been developed 
which indicate that abnormal stress distribution 
within the structure of the lung may be an impor- 
tant factor in the production of the disruptive 
lesions of emphysema. Using an improved intra- 
esophageal pressure measuring system, the 
intrathoracic pressure has been measured simul- 
taneously at three different sites along the 
esophagus. The differences between oral pres- 
sure and the intraesophageal pressures have been 
studied under static conditions in several normal 
subjects. It has been possible to describe this 
relationship as a function of balloon volume, 
balloon position, and lung volume. The relation- 
ship seems to be independent of the rate of change 
of pressure or of the amplitude of pressure ' 
changes. This initial part of the project, which 
is nearing completion, is preliminary to studying 
the pressure differences among the three balloons 
during conditions of air flow, particularly during 
cough. If it can be shown that abnormal stress 
distribution occurs in subjects known to have a 
high incidence of pulmonary emphysema, indirect 
evidence in support of the foregoing theory will 
be obtained. 

Theoretical considerations based on anatomical 
and biophysical studies of excised bronchi indicate 
that the relationship between the maximum ex- 
piratory flow at any given degree of lung inflation 
should be controlled by the density and viscosity 
of the gas breathed as well as the dimensions and 
physical properties of the intrathoracic airways. 
If it can be shown experimentally that tlie effect 
of either density or viscosity on this relationship 
causes changes in the flow-volume relationship 
which would be predicted by the equations de- 
scribing the theory, evidence in support of the 
theory would be gained. The findings thus far 
are in general agreement with the theory; how- 
ever, computational methods must be developed to 
quantify the degree of agreement. 

SURGERY BRANCH 

The investigative projects of the Surgery 
Branch have, as in past years, largely centered 



NATIONAL HEART INSTITUTE 



129 



around the development of new methods for the 
surgical treatment of patients with congenital or 
acquired heart disease. In addition, the majority 
of projects carried out in the Experimental Sur- 
gery Laboratory have been designed to elucidate 
the physiologic changes associated with reproduc- 
tion, in experimental animals, of various forms of 
heart disease and of the operations designed to cor- 
rect them. The results of the experimental work 
have been applied in the continuing clinical pro- 
gram in which opportunity has been taken to 
obtain physiologic measurements in the course of 
both open and closed cardiac operations. 

Aneurysms of the left ventricle, when seen in 
patients, almost invariably have resulted from 
coronary atherosclerosis and a subsequent myo- 
cardial infarction. Under these circumstances, 
the hemodynamic effects of the aneurysm are dif- 
ficult to distinguish from those of the underlying 
myocardial disease. To determine the hemo- 
dynamic effects of left ventricular aneurysms on 
the normal circulation they were created in dogs 
attaching an excised urinary bladder to the left 
ventricle and creating a communication between 
this sac and the ventricular cavity. Effective left 
ventricular function and cardiac output were de- 
termined in these animals when the aneurysm was 
open and closed. It was found that significant 
acute depressions in effective left ventricular func- 
tion and in forward cardiac output occurred when 
the aneurysm was opened. 

In many open operations for the correction of 
both the tetralogy of Fallot and pulmonic stenosis 
it is necessary to induce pulmonary regurgitation 
and sometimes to enlarge the outflow tract of the 
right ventricle with a plastic prosthesis. The 
physiologic effects of various surgical procedures 
on the pulmonic valve and right ventricle were 
evaluated. Effective right ventricular function 
was only slightly depressed when the right ven- 
tricle was incised. When pulmonic regurgitation 
was produced by any means, however, additional 
impairment of function occurred and total exci- 
sion of the valve usually resulted in such severe 
depression of function that the curve could not 
be inscribed. Following these operations a more 
detailed study of the physiologic effects of pul- 
monic regurgitation was undertaken in which the 
volume of regurgitant blood flow was measured, 
either by an electromagnetic flowmeter or by the 



application of indicator dilution methods. The 
excision of one pulmonic valve leaflet was found 
to cause a regurgitant flow equal to about half 
of net forward cardiac output and in experiments 
in which two or three leaflets were removed regur- 
gitant flows in excess of forward output were 
measured. When allowance was made for the 
volume of regurgitant flow in the stroke volume 
of the ventricle it was found that true or corrected 
ventricular function was impaired by these 
valve lesions, but that effective ventricular func- 
tion was severely impaired. The results of these 
studies will have immediate clinical application in 
the operative treatment of patients with obstruc- 
tion to right ventricular outflow. 

In the treatment of a variety of congenital le- 
sions it is necessary to increase pulmonary blood 
flow and this is usually accomplished by the crea- 
tion of a systemic to pulmonary artery shunt. In 
the conventional Blalock operation, in which the 
left subclavian artery is anastomosed to the pul- 
monary artery, it has been observed that as the 
patient grows the shunt presumably stays of the 
same magnitude and with increasing age the bene- 
fit derived by the patient from the procedure 
diminishes. A number of Russian surgeons have 
modified the Blalock operation and construct the 
anastomosis by means of a large vascular graft in- 
serted between the side of the subclavian artery 
and the side of the pulmonary artery. Thus, the 
size of the shunt is not limited by the size of the 
anastomoses but by the size of the subclavian 
artery, and, on clinical grounds, the Russians 
believe that as an infant or young child grows the 
size of the shunt and consequently the increase in 
pulmonary blood flow will remain relatively con- 
stant. In order to test this hypothesis, the Russian 
operation is being carried out in newborn puppies 
and in newlborn lambs. The relationship of shunt 
flow to total cardiac output is being measured in 
the animals with growth. In this anastomotic 
operation, as well as those carried out for the 
reconstruction of obliterative arterial disease, 
vasopressor agents are frequently used in the im- 
mediate postoperative period either to counteract 
hypotension or to increase flow througli the anas- 
tomosis or prosthetic artery. A detailed study is 
in progress in which blood flow in the aorta and 
various peripheral arteries is measured by means 
of an electromagnetic flowmeter and the effects on 



130 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



regional blood flow of various vasopressor drugs is 
being evaluated. The preliminary results of these 
studies at this time, contrary to clinical impres- 
sion, mdicate that the majority of pressor agents, 
although raising blood pressure, cause a fall in 
systemic arterial flow. 

Generalized or local cardiac hypothermia is be- 
ing employed throughout the country with increas- 
ing frequency in conjunction with open cardio- 
vascular operations. A number of projects in the 
past year have dealt with physiologic studies of 
hj'pothermia. In dogs previously subjected to to- 
tal cardiac denervation, it was found that there 
was a far more striking fall in the heart rate with 
cooling than in normal animals and when dener- 
vated dogs were subjected to acute hemorrhage no 
significant change in the heart rate or contractile 
force of the ventricle occurred. In an extension 
of this work normal dogs were subjected to acute 
hemorrhagic shock as contractile force was meas- 
ured. It was found that down to an arterial pres- 
sure level of 90 mm. Hg contractile force was un- 
impaired, but below this pressure level a rapid 
decrease in heart rate and a decline in contractile 
force occurred. These, and the previous observa- 
tions, indicated that both neurogenic and intrinsic 
cardiac eilects are operative in the early stages of 
acute hemorrhagic shock. The observations were 
then extended in normal dogs in which the sys- 
temic and coronary arterial circulations could be 
perfused separately by two extracorporeal sys- 
tems. In these animals it was possible to "shock" 
all of the body except the heart in which normal 
flow and pressure were maintained. In the first 
stage of acute systemic hypotension the heart rate 
and contractile force increased slightly after 
which there was a steady gradual decline in both 
measurements. Thus, all of the above observa- 
tions indicate that, during early acute hypovo- 
lemic shock, para-sympathetic stimulation occurs 
and primary cardiac effects are not evident at this 
time. With prolongation of the shock state, how- 
ever, a second mechanism, probably a humoral 
one becomes opei-ative and is responsible for the 
decline in cardiac efficiency. 

Surgical opinion remains varied as to the best 
means for maintaining the functional integrity 
of the heart following long periods of aortic oc- 
clusion, such as are necessary in partial or total 
replacement of the aortic valve. A comparative 



study was carried out to determine the technic 
which would minimize impairment of ventricular 
function after aortic occlusion. As found previ- 
ously, simple cardiopulmonary bypass did not de- 
press ventricular function and aortic occlusion 
and anoxic arrest could be tolerated by the normal 
dog for only 20 minutes. Intermitten perfusion of 
the coronaries with warm oxygenated blood im- 
proved subsequent function but marked depression 
usually resulted nevertheless. When the heart 
was first rendered hypothermic, anoxic arrest was 
better tolerated. However, only when local lij'po- 
thermia of the heart was supplemented with in- 
termittent coronary perfusion was satisfactory 
subsequent function indicated in the fimction 
curves. In spite of the results of these experi- 
ments, it is still felt that insufficient information 
is available concerning the effects of hypothermia 
on the heart and local cardiac hypothermia is not 
employed in patients. 

When the heart is cooled, by any means, it has 
long been recognized that the rate slows and an 
increase in ventricular contractile force occurs. 
In order to determine whether this response was 
due to the direct cardiac effects of cooling or was 
the result of neurogenic stimulation, expei-iments 
were carried out in which the temperature of tlie 
heart and the remainder of the body could be in- 
dependently varied during extracorporeal circula- 
tion. Initial experiments indicate that when the 
body is cooled and the heart remains normo- 
thermic that little change in its rate or strengtli 
of contraction occurs, indicating that the effect is 
in all likelihood a local rather than a central one. 

Extreme degrees of hypothermia are sometimes 
applied when it is necessary to interrupt the cir- 
culation completely during the repair of paiticu- 
larly complex intracardiac anomalies. Wlien the 
temperature is lowered to profoundly hypothermic 
levels (12-15°C.) it has been postulated by others 
that the hemoglobin dissociation curve is shifted 
so far to the left that oxygen may not be available 
to the tissue from oxygenated hemoglobin. On 
this basis numerous surgeons have advocated that 
acidosis be induced during cooling to shift the 
curve toward a more nonnal position. Acidosis is 
known to have deleterious effects of many types on 
the circulation and an expei'imental investigation 
of the desirability of acidosis during hypothermia 
was undertaken. Dogs were cooled to 12°C. and 



NATIONAL HEART INSTITUTE 



131 



in some the blood. pH was lowered during cooling 
by the administration of HCL and in others it was 
raised by the administration of bicarbonate. The 
total body oxygen consumption was unaltered by 
these changes in blood pH indicating that the 
shift in the hemoglobin dissociation curve is of 
little importance at these temperatures. The aortas 
of these dogs were occluded for 30 minutes after 
they had been rendered hypothermic and either 
alkalotic or acidotic. Function curves were then 
inscribed after the animals had been rewarmed. 
It was found that the dogs rendered acidotic 
showed severe depression of subsequent ventricular 
function, as did dogs with a normal pH, but that 
dogs previously rendered alkalotic had distinctly 
better ventricular function. It was concluded that 
in clinical practice acidosis during hypothermia 
serves no useful purpose and, since it has a delete- 
rious effect on the heart, it is probably undesirable. 

In the large nimiber of open operations that have 
been carried out at the National Heart Institute, 
it has been our clinical observation that following 
cardiopulmonary bypass patients often require 
supplemental digitalis in the first 24 hours. To de- 
termine the effect of cardiopulmonary bypass on 
the digoxin content of the heart a supply of tri- 
tium labeled digoxin was obtained and given to 
normal dogs. Biopsies of the heart and other tis- 
sues were obtained and the animals were then 
subjected to 30 minutes to cardiopulmonai'y bypass 
after which biopsies and blood samples were again 
obtained and analyzed for radioactivity. It was 
found that 30 minutes of cardioj)ulmonary bypass 
caused the heart to lose approximately 14 of its ra- 
dioactivity and that the radioactivity of the blood 
in the dog and heart lung machine rose strikingly. 
By means of paper chromatography it was shown 
that at least i^ of the radioactivity which left the 
heart was unchanged digoxin and the remainder 
presmnably its metabolic products. By using 
tagged digoxin with a higher specific activity pre- 
liminary obseiwations have been made in seven 
patients subjected to open operation and similar 
decreases in radioactivity of the heart have been 
observed. Continuing studies on the mechanism 
by which digoxin is lost from the heart are being 
carried out in both patients and animals. 

In the past year much progress has been made 
throughout the country in the problem of pros- 
thetic replacement of the aortic and mitral valve. 



Following the initial encouragmg results with 
the foam plastic mitral valve developed here, its 
use has been abandoned until further refinements 
can be made. Modifications have been made in the 
configuration of the artificial leaflets and the 
method of attachmg the artificial chordae ten- 
dineae to them. The modified valve is presently 
being tested in experimental animals to determine 
its hemodynamic effects and in an attempt to ob- 
tain chronic survival. The same plastic molding 
technics which were developed in the design and 
fabrication of the prosthetic mitral valve are now 
being applied in the development of prosthetic 
total aortic valves and individual aortic valve 
leaflets. The total prosthesis has been constructed 
in a size suitable for implantation into the descend- 
ing thoracic aorta of dogs and is now being eval- 
uated in this anatomic position in animals in which 
aortic regnirgitation has been produced. Informa- 
tion concerning thrombosis and ultimate mobility 
of the prosthesis will be obtained before attempts 
at insertion in the subcoronary position are made. 

Prosthetic mitral or aortic valves must be con- 
structed of some plastic or metal substance and 
work continues on the evaluation of various pros- 
thetic materials in the circulation, with particular 
regard to clotting. Dacron cloth has been coated 
with polyurethane foam with both open and closed 
cell surfaces. Pieces of the material have been 
implanted into the wall of the heart and also sus- 
pended within the cavities of the heart by means 
of artificial chordae tendineae of various types. 
Chronic preparations of this type over a period 
of a year or more will allow a comparative evalu- 
ation of the surface which is most desirable and 
the material most suitable for the suspending 
chordae tendineae. Preliminary observations in- 
dicate that substances with closed cell surfaces are 
more desirable but they do not, of course, allow 
for tissue ingrowth. Attachment of the chordae 
tendineae to the wall of the ventricle by means of 
aluminum buttons has been f omid to be satisfac- 
tory even when extreme tension has been placed 
upon them. Chordae of teflon or polyurethane 
coated silk apparently function better than those 
of wire or other substances. 

An outgrowth of the work on prosthetic valves 
has been the discovery that a plastic adhesive 
(Eastman 910 monomer) is a useful adjunct in 
both experimental and clinical cardiovascular 



132 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



surgery. This agent is a rapidly polymerizing 
material which adheres with ease to dry living 
tissues and causes little undesirable foreign body 
reaction. It was evaluated as a method of con- 
trolling hemorrhage from the heart and great ves- 
sels. Incisions were made in the heart or in the 
aorta and, utilizing the adhesive, a patch of pros- 
thetic material or skeletal muscle was cemented 
over the site of injury. Satisfactory hemostatis 
was obtained by this method in virtually all ex- 
perimental animals, but before clinical applica- 
tion is extensive it will be necessary to obtain 
further long term information about the ultimate 
histopathologic fate of the material. The mon- 
omer solidifies almost instantly on contact with 
blood and when it was accidentally injected into 
the vascular tree it was found to promote wide- 
spread vascular clotting. This suggested that oc- 
clusive lesions of the arterial or venous system 
could be experimentally produced by selective 
injections of the monomer. This has proved true. 
With left atrial injection complete occlusion of 
the aorta could be produced as well as occlusions 
of the coronary, renal, celiac or femoral arteries. 
Similarly, by injection of the monomer into the 
pulmonary artery or the portal vein, occlusive 
lesions resulting in pulmonary or portal hyper- 
tension were easily produced. It would seem that 
this technic may prove to be a valuable one in the 
experimental production of various occlusive ar- 
terial lesions. 

Immediately after the correction of an intra- 
cardiac lesion acute right or left sided heart failure 
may supervene and be responsible for early post- 
operative death. Under these circumstances it 
would be desirable to be able to support the 
circulation by some mechanical means until the 
heart was able to recover from the acute trauma 
of the operation and again support full cardio- 
vascular function. Various investigators have 
demonstrated that the work and oxygen re- 
quirements of the failing heart may be de- 
creased by partial extracorporeal circulation 
but partial bypass has almost invariably resulted 
in severe metabolic acidosis. This finding has been 
confirmed in laboratory experiments on partial 
cardiopulmonary bypass for periods of an hour 
or more. In addition, abdominal distension and 
engorgement of the abdominal viscera were noted. 
In the system evaluated no oxygenator was em- 



ployed and venous blood was returned through a 
pump to the femoral artery of the animal. It 
seems likely that the "pooling" of blood within 
the abdomen may be related to the fact that un- 
oxygenated blood is delivered to the lower half 
of the body from the perfusion system and this 
has been confirmed by arterial oxygen determina- 
tions in the descending aoita. Continuing work 
on this important adjunct to cardiovascular sur- 
gery and the role that THAM and other buffer 
agents may play in the amelioration of the acidosis 
is underway. 

The orthopedic resident assigned to the Surgery 
Branch has continued collaborative work with the 
National Institute of Arthritis and Metabolic 
Diseases. The hyaline sclerosis of synovial blood 
vessels described in the previous report has been 
subjected to further study. This heretofore un- 
described pathologic lesion was found in the vast 
majority of all normal joints studied. The sig- 
nificance of the lesion remains unknown and con- 
tinuing investigations of the etiology and possible 
significance of the finding are underway. The 
osseous changes resulting from arterial oxygen 
desaturation are well known clinically but little 
information is available concerning the method 
by which the pathologic bone changes develop. 
Arterial oxygen unsaturation has been produced 
in dogs by various surgical procedures, such as 
pulmonary arterial-left atrial fistula and pulmo- 
nary arterio-venous fistula. Arterial oxygen 
saturations as low as 50 percent have been obtained 
in chronically surviving animals and they are 
being subjected to detailed radiographic study. 
Inequality in the length of long bones is a common 
and distressing orthopedic problem, particularly 
in children. Many operations to stimulate bone 
growth selectively have been evaluated but none 
has stimulated sustained growth sufficiently to 
totally equalize limb length. For this reason the 
best clinical results have been obtained from oper- 
ations in which bone growth was retarded in the 
normal limb. A new mechanical method for stim- 
ulating bone growth is being evaluated. A pair 
of compressed coil springs, anchored to a metal 
bar, are attached to the diaphysis in such a manner 
that there is constant tension between the diaphysis 
and the growing epiphysis. The effectiveness of 
this method of stimulating bone growth is being 
evaluated in growing puppies. Chemical adhesives 



NATIONAL HEART INSTITUTE 



133 



have also been found of interest in the orthopedic 
field and two experimental polymers were eval- 
uated in the repair of osteotomies in dogs. Path- 
ologic studies revealed that the compounds were 
unsatisfactory for this purpose since they were 
exothermic in tissue, causing burns, and did not 
bond sufficiently well to bone to permit stabiliza- 
tion of a fracture unless they were supplemented 
with metal support. Neither was found as effec- 
tive as polyurethane foam for this application. 

GERONTOLOGY BRANCH 

The research program of the Gerontology 
Branch is directed toward (1) describing the bio- 
chemical, physiological and psychological changes 
that take place with increasing age in man, and 
(2) investigating the basic biological changes that 
contribute to aging in order to understand age- 
dependent alterations in the performance of hu- 
mans. During the past year, primary emphasis 
has been placed on investigations of basic bio- 
chemical processes which are essential in main- 
taining life in the cell. 

In this connection the enzymatic reactions as- 
sociated with the utilization of oxidative energy 
for the synthesis of high energy compounds like 
ATP for subsequent use in muscle contraction and 
synthesis of tissue constituents have been investi- 
gated. The work carried out during the previous 
year indicated that the key reaction in the energy 
trapping system in mitochondrial electron trans- 
port may involve a dithiol grouping. The current 
work on this problem has established with reason- 
able certainty that the system does indeed contain 
a functional dithiol group. The uncoupling of 
ATP synthesis from oxidations by arsenite under 
well-defined conditions has been observed now in 
mitochondrial fragments prepared from rat liver 
and beef heart mitochondria. The physical 
changes in mitochondria (swelling) in the pres- 
ence of the uncoupling agent have been shown to 
occur subsequent to the uncoupling. 

The oxidative decarboxylation of a-ketogluta- 
rate to succinyl CoA is another reaction in which 
the oxidative energy is used for ATP synthesis. 
This system has two functional dithiol com- 
pounds — the dihydrolipoate associated with the 
primary oxidation of the aldehyde to the level of 
the active acyl and the unidentified dithiol com- 



pound present in the highly purified dihydroli- 
poyldehydrogenase flavoprotein. The participa- 
tion of the latter dithiol, previously inferred from 
inhibition of the enzymatic activity by arsenite, 
has been established by direct titration with 
p-chloromercuribenzoate. 

The results clearly indicate that dithiols have 
unsuspected key roles in oxidative and energy 
trapping reactions, and have opened up a possibly 
rewarding approach to the study of the mecha- 
nism of oxidative phosphorylation. 

Since developmental processes set the stage for 
senescent changes investigations on cellular 
changes during the process of maturation have 
been carried out. The developmental studies are 
designed to test the hypothesis that : 

(a) A continuation of developmental proc- 
esses beyond functional maturity may lead to 
less viable rather than more viable cells. 

(b) The loss of developmental plasticity 
characteristic of cells during normal develop- 
ment continues during later maturation and 
senescence. 

Evidence obtained during the past year has con- 
firmed that the formation of muscle syncytia in 
tissue culture as reported earlier from this lab- 
oratory occurs by cellular fusion rather than by 
nuclear division unaccompanied by cytoplasmic 
division. This fact was shown both by time-lapse 
photomicrographic studies and by the measure- 
ment of DNA in syncytial nuclei and mononu- 
cleate cells using a specially built and designed 
microspectrophotometer. All syncytial nuclei 
were found to be diploid in their DNA content, 
whereas mononucleate cells were found to contain 
diploid, tetraploid and intermediate quantities of 
DNA. 

The kinetics of the differentiation process in 
muscle syncytia have also been followed by meas- 
uring the concentration of creatine-kinase in such 
■preparations at various stages of development as 
well as in vivo. The concentration rises in a 
manner characteristic of an autocatalytic reaction 
over a considerable portion of the period of dif- 
ferentiation. More recently it has become possible 
to inhibit or accelerate the differentiation process 
by control of specific environmental factors. 

The effect of age on the capacity of human mus- 
cle explants to give rise to new outgrowths in tissue 
culture is being examined. Successful culture ap- 



134 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



pears feasible using present metliods only in a 
small fraction of cases, but the important varia- 
bles differentiating successive experiments liave 
not yet been worked out. 

Studies on the senescent phase of cell life have 
concentrated heavily on the chemical nature of 
the so-called lipofuscin age pigment of human 
cardiac tissue and the possibly related inclusion 
bodies occurring in the short lived experimental 
animal, Gampanularia fiexuosa. Improved meth- 
ods of isolating age pigment in highly concen- 
trated and purified form from human myocardium 
have been developed. The preparations obtained 
have been subjected to systematic examination by 
infra-red analysis, fluorometry, microspectro- 
photometry, amino acid and elementary analysis 
as well as silicic acid and paper chromatography 
of the constituent lipids and gas chromatography 
of the fatty acids derived from methanolysis of 
chromatographic fractions. Two major fluores- 
cent bands are typically obtained from chromatog- 
raphy of the lipid soluble components. These 
bands are eluted from silicic acid columns along 
with the cardiolipin and cephalin fractions. The 
residue remaining after extraction with lipid sol- 
vents yields free amino acids and an insoluble 
residue upon hydrolysis in either base or acid. 
This residue contains further lipid soluble pig- 
ments and an insoluble component. Enzymati- 
cally, such prej)arations are essentially inactive, 
containing only minor amounts of either the lytic 
enzymes associated with lysosomes or of the 
respiratory enzymes of mitochondria. This 
absence of lysosomal enzymes is not consistent 
with results obtained using less drastic methods 
of preparing age pigment (an early part of this 
present procedure is sonication). Since histo- 
chemical studies by Gedigk and Bontke and by 
Essner and Novikoff had indicated a close associa- 
tion between lif)ofuscin and lysosomal enzymes, 
the low activities found in the most purified pig- 
ment preparations suggest either that the pigment 
of aged human myocardium is no longer associated 
with lysosomal enzymes or that treatment during 
isolation has liberated j^reviously associated 
enzymes. 

The occurrence of increasing numbers of acid 
phosphatase positive granules in aging Oampanu- 
laria suggests that the normal senescence and 
death of individuals of this species occurs through 



the action of lysosomal enzymes. The histo- 
chemical studies on cardiac lipofuscin referred to 
above furnish a link between the "primitive" 
senescence of Oampanuhina and that occurring in 
human fixed postmitotic cells. 

Estimates of cathepsin activity have been made 
on various fractions of cell particulates separated 
by differential centrifugation and density gradi- 
ents. Catheptic activity in percent of total re- 
coverable activity is distributed as follows: 
nuclei, 10% ; mitochondria, 36% ; lysosomes, 35% ; 
supernatant, 19%. 

Stabilized cultures of Euglena cells have been 
used as a model to study age effects. The results 
so far show that Euglena. cells can survive for 
about 12 days without any external source of car- 
bon or energy and without loss of viability. Dur- 
ing this time the cell consumes most of its 
glycogenstarch reserves, half of its protein and 
half of its RNA, its DNA appears to become 
haploid, and its oxygen consuming capacity be- 
comes exceedingly small. The content of carot- 
enoids, however, does not decrease demonstrating 
that not all components of the cell are available 
for utilization. The time course of resynthesis 
of these components has also been determined 
when the cells are resupplied with acetate after 
5, 8, and 12 days of starvation. Oxygen consump- 
tion capacity rises very sharply before any net 
synthesis of protein occurs. This points to the 
existence of a control mechanism in this cell and 
we intend to examine the cell for the nature of 
this mechanism (feed back or enzyme repression 
or both?). The system also is of interest since 
it provides a nondividing culture in which the re- 
synthesis of 50% of the ENA and protein of tlie 
cell occurs, and hence the system can be used for 
studying some aspects of ribosome formation. 

Work has also progressed on the study of the 
synchronized division of Astasia. The system has 
been improved so that division occurs in a rather 
reproducible fashion every day, and with suffi- 
cient rapidity so that the culture actually exists 
in the haploid state for a short time during tlie 
peak of mitotic activity. It has been found that 
8-azaguanine, an analogue of guanine that was 
originally used for blocking nucleic acid synthesis, 
has an effect on the synchronized system, pro-\'ided 
it is present in tlie cold period, but lias little effect 
on logarithmically growing cultures. Tlius we 



1 



NATIONAL HEART INSTITUTE 



135 



liaA^e demonstrated the existence of a key reaction 
in the cold period which is essential to the timing 
of mitosis. Present evidence stronglj^ suggests 
that the 8-azagua.nine is not interfering with nu- 
cleic acid synthesis, and we intend to pursue this 
study and attempt to characterize the process that 
is being blocked by 8-azaguanine. 

In the field of protein studies, we have finished 
a project on actomj'osin aimed at elucidating some 
of the relations between the role of the sulphydryl 
groups in ATPase activity and their role in con- 
tractility. When small amounts of certain — SH 
reagents react with actomyosin, the ATPase activ- 
ity is greatly enhanced. We have shown that 
when this happens, contractility is not interfered 
with and, furthermore, the ability of a relaxing- 
factor system to cause relaxation is also not inter- 
fered with. Thus the first — SH gi-oup to react 
is not essential for contraction or relaxation, al- 
though it exerts a considerable influence on 
ATPase activity. Further treatment with — SH 
reagents reverees the activation of ATPase activ- 
ity and it is found that contractility is lost before 
there is any net inhibition of the ATPase. This 
suggests that the integritj^ of mechano-chemical 
coupling requires the integrity of a particular 
— SH group on the myosin and does not depend 
on the binding of ATP or the overall rate of 
hydrolysis of ATP. 

A cold-precipitable protein obtained from a pa- 
tient whose diagnosis was essential cryoglobuli- 
nemia has been studied. Samples of blood were 
obtained from the patient's wife, daughter, sons 
and grandchildren. Two of the sons had a crj^o- 
globulin in their blood serum. The protems were 
partially characterized by ultracentrifugation, 
paper electrophoresis and amino acid analysis. 
The evidence suggests the possibility that cryo- 
globulinemia is an inherited molecular disease. 

The most important project of the Section on 
ISIolecular Biology at the present time is the one 
designed to study the nature of the reaction of 
metal ions and other substances in the nucleic acids 
and their derivatives. The ultimate goal is to 
provide a chemical method for the determination 
of sequences of nucleotides in nucleic acids, a goal 
of great importance because this sequence is be- 
lieved to constitute the coding of the hereditary 
information of the cell. The studies are of interest 
pei^ se, however, since they provide clues to the way 



in which the structure of complicated macromole- 
cules is regulated by the interactions with small 
substances such as metal ions. 

Two questions are of prunary importance in an 
investigation of the binding of metal ions to nu- 
cleic acids. (1) Do the metals bmd to the outside 
of the molecule, neutralizing the charge on the 
phosphates, or to the inside coordinating to the 
hydrogen-bonded bases? (2) If the metals bind 
to the inside, do thej' bind equally to the four 
nucleotides, or do they diiferentiate among them ? 

The first question has been answered for a large 
nmnber of metal ions; some bind to the outside, 
and some to the inside. Binding to the outside is 
important because stabilization of the molecule is 
accomplished ; the metals that bind in this way can 
be ruled out in sequence determination studies. 
Binding to the inside means competition with 
hydrogen bonding and consequent weakening of 
the macrostructure. 

The second question is more difficult to answer 
because each metal ion seems to react in a different 
manner. The question is important to sequence 
determination studies, since a reaction is useful in 
such a determination only if it can pick out one of 
the four code-producing nucleotide bases, and 
leave the other three alone. 

The summation of the many studies that have 
been carried out is that metals do differentiate 
among the nucleotides, but in general the differen- 
tiation is quantitative, rather than qualitative. 
Nevertheless, by manipulating conditions, such as 
pH, a metal can be made to prefer one of the four 
nucleotides very substantially over the others. It 
has been possible to obtain a great deal of specific- 
ity in a number of cases. 

The second project, that of the function of metal 
ions in enzj'matic reactions, has led to the further 
elucidation of two reaction mechanisms. In both 
cases, the same question has been answered in the 
same way. The question : Does the metal function 
to bring enzyme or coenzyme in contact with sub- 
strate, or does it function as the catalytic site for 
the reaction? The answer: the latter. Studies 
on models for the vitamin Be catal^yzed reactions 
have shown that metals actually tend to prevent 
contact between coenzyme and substrate. Studies 
on the reaction of metal with the aconitase sub- 
strate indicate that the metal is the prime agent 



136 



ANNUAL REVIEW OF ESTTRAMURAL RESEARCH 



responsible for the essence of the reaction ; namely, 
the removal of a hydroxyl group for citric acid. 
During the past year efforts have been made to 
identify age changes in terms of various biochem- 
ical and physiological parameters and to determine 
the extent to which these changes may be explained 
on the basis of loss of cells. These experiments 
have been performed on adult and senescent rats. 

It was shown that, except for a greater loss of 
muscle mass, the senescent loss in organ weights 
and changes in enzymatic activities observed in 
the tissues of '34 as compared to 14 month old wild 
rats were similar to, but of no greater magnitude 
than, those detected when 12 and 24 month old 
domesticated rats were examined. 

In other experiments, designed to estimate the 
maximal transport of PAH by kidney slices, no age 
differences were found in the initial rate of ac- 
cumulation of PAH (yiig PAH/gm wet weight — 
fig PAH/ml. medium after 15 minutes incubation) . 
However, the maximum accumulations {fj.g PAH/ 
gm wet weight — /xg PAH/ml medium after 60 
minutes) observed in 14 and 30 month old rats were 
18% and 39% lower respectively than that found 
in 3 month old animals. Since these decrements 
exceeded the agewise reduction in the number of 
cells, as estimated by the concentration of DNA, 
these data suggest that aging is accompanied by 
an alteration in the function of proximal tubule 
cells, assuming that there is no selective loss of 
tubule cells from the total population of renal 
cells. 

Other studies were carried out to estimate age 
changes which occur in collagen by measuring the 
rate of change in the chemical contraction and 
relaxation of rat tail tendon fibers immersed in 
2.5 M sodium perchlorate. Although differences 
of 30^0% were found between young growing and 
mature rats, differences of only 5-10% were ob- 
served from maturity to 2 years, after which no 
changes were evident. 

Other data failed to demonstrate any differ- 
ences in the response to atherogenic diets as esti- 
mated by changes in the concentrations of liver 
succinoxidase, plasma or liver cholesterol or in the 
extent of vascular sudanophilia when 12 and 24 
month old male rats were compared. However, 
the vascular sudanophilia was less in young grow- 
ing animals than in the old. 



As an attempt to test the hyjjothesis that re- 
duced dietary intake results in increased life span 
by prolonging the growth phase, experiments were 
carried out to compare some biochemical and phys- 
iological characteristics in normal animals and 
those subjected to caloric restriction of the diet. 

An increased concentration of succinoxidase in 
liver and of alkaline phosphatase in kidney was ob- 
served in weanling male rates offered 50% of the 
amount of diet consumed by ad lib. fed animals for 
periods of 5 weeks to 10 months. In female ani- 
mals, however, the increase in the latter enzyme 
was not evident. Adult female rates, so restricted 
for 8 weeks, showed the same pattern of change 
observed in young growing females subjected to 
dietary restriction. These data fail to support the 
concept that dietary restriction prolongs the grow- 
ing phase in animals, since the reduced intake re- 
sulted in higher concentrations of selected enzymes 
which are low in chronologically younger animals. 
The similarity in response to dietary restriction in 
adult rats and young growing animals suggests 
that life span may be increased by a reduced 
dietary intake in adult animals. Thus, retarda- 
tion of normal growth processes may not be neces- 
sary for increased longevity. 

However, measurements of the chemical contrac- 
tion and relaxation of fibers isolated from the tail 
tendons of both male and female restricted rats 
were similar to chronologically younger animals. 
Estimates of the total activity of ad lib. fed and 
restricted rats made in the suspension type cage 
demonstrated a lower activity in restricted rats. 
In the wheel cage, where an increased activity due 
to hunger drive was found bj' a comparison of ad 
lib. fed animals and acutely restricted rats of the 
same age, rats chi'onically restricted during growth 
had a lower activity. At present, it is not possible 
to evaluate the latter findings in terms of growth 
retardation due to incomplete data regarding the 
effect of body weight on activity measurements in 
the suspension cage and the quantitative aspect of 
the hunger drive in terms of wheel running. 

Taken as a whole, these data do not completely 
support the concept that reduced dietary intake 
results in a retardation of the temporal sequence 
of changes accompanying normal growth and 
thereby increases life span. 

A method for determining lung compliance in 
the intact human has been developed and standard- 



NATIONAL HEART INSTITUTE 



137 



ized. Data on the effects of aging on this param- 
eter are being collected. 

A method for the quantitative estimation of 
erythrocyte agglutinability has been developed and 
a study of the agglutinability of B type cells from 
old and young donors is in progress. This study is 
aimed at determining whether aging is associated 
with a change in the synthetic processes involved in 
the formation of red cells. Measurements of the 
effects of epinephrine and norepinephrine on pul- 
monary blood volume in anesthetized dogs (closed- 
chest) have been completed. Intravenous infu- 
sion of these drugs in doses ranging from 0.5 to 2.0 
/*g/kg/min increased pulmonary blood volume 
significantly. A simultaneous and proportional 
increase in the effective pulmonary artery and left 
atrial pressures suggested that the increase in pul- 
monary blood volume was predominately passive. 
Since these drugs have been shown to have an ac- 
tive, direct, constrictor effect on some pulmonary 
blood vessels, the observed response in the intact 
dog appears to be an example of passive distention 
outweighing the effects of active constriction. 

Infusion of isoproterenol (2.0 /xg/kg/min) and 
of histamine (5 jug/kg/min) also increased pul- 
monary blood volume, with either no change or a 
decrease in transmural vascular pressure. This 
suggests that these drugs cause an intrinsic dila- 
tion of some part of the pulmonary vascular bed, 
larger in volume than the arterioles alone. 

Investigation of the effects of negative pressure 
breathing on pulmonary blood volume in man has 
given inconclusive results. Further studies in 
which both extra-thoracic and air-way pressures 
are controlled will be carried out. 

Investigation of the effects of negative pressure 
breathing on pulmonary blood volume in man has 
given inconclusive results. Further studies in 
which both extra-thoraric and air-way pressures 
are controlled will be carried out. 

The rate of disappearance of the hemoglobin- 
haptoglobin complex from the plasma was linear 
with time in 15 subjects. The rate of disappear- 
ance from the blood was independent of the level 
of the complex in the plasma. 

Urine osmolality of samples collected at 6:00 
p.m. in subjects permitted their usual water intake 
diminished from 950 milliosmols/L in subjects 
under 40 years of age to 850 milliosmols/L in sub- 
jects over 70. Wlien no fluid intake was per- 



mitted after 6:00 p.m., the young subjects 
showed an increase in urine osmolality by 
12:00 m., whereas there was a slight decrease in 
osmolality in the old subjects. This differential 
response of old and young subjects may be due to 
the fact that the kidneys of the old subjects are 
operating near their maximum concentration ca- 
pacity under most circumstances or that the tu- 
bules of the aged kidney are less sensitive to ADH 
or that the old subjects liberate less ADH follow- 
ing the mild fluid deprivation than do the young. 
Further experiments are planned. 

The pattern of solute excretion during the night 
also shows age differences. The youngest sub- 
jects excreted 60% of their total 14 hours solute 
excretion in the first 6 hours (6 :00 p.m.-12 :00 m.), 
whereas the old subjects excreted only 45% of the 
total during this time. 

Two independent estimates of body fat have 
been compared in a group of 100 subjects. An 
equation using our estimates of total body density 
and of age reduction in creatinine excretion and 
bone density estimates a decrease in fat in older 
subjects which averages 0.12% fat per year. An 
anthropometric index which summarizes the ma- 
jor circumferences and diameters of the body esti- 
mates a decrease in fat in older persons which 
averages 0.2% fat per year. Thus, the lower 
weight foimd in these older persons represents a 
lesser amount of both fat and lean tissue. 

Preliminary results of a sensory-motor test of 
tapping between similar targets for speed and ac- 
curacy indicate that persons over the age of 60 
take additional time to perform the tasks involved 
and that the slope of the curve relating movement 
time to movement distance and target width is 
different for old and young. The relation between 
movement time and overall information transfer 

/ , ^. effective movement distanceX 

I movement time = r — ^j-, I 

\ target width / 

gives a curve for persons under 60 years of age 
which must be corrected by adding time for errors, 
in order to extrapolate to zero time for zero infor- 
mation transfer. In persons 60 years of age and 
over, additional time for all tapping tasks results 
in a residual time of 0.05 to 0.10 seconds at zero in- 
formation transfer when the curves of the move- 
ment time, information transfer relationship is 
extrapolated to zero information transfer. These 



I 



138 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



residuals can only be increased by correction for 
errors. Tims the "increased times for all tasks" 
does not lend itself to the interpretation that in- 
creased difficulty of a task alone places the older 
person at a relatively greater disadvantage but 
rather that some factor -which works at all levels 
of difficulty is responsible for part of the addi- 
tional time taken by the old. 

Limg volume and maximum breathing capacity 
measurements were compared between an indigent 
group in an old jseoples home and a self-support- 
ing community residing group. Even when indi- 
viduals with clinical ratings of moderate or 
marked ventilatory functional impairment were 
excluded from the comparisons, differences were 
found between the two groups. The community 
residing group (50-69 years old) had significantly 
(P=<.001) better maintained vital capacity and 
maximum breathing capacity than the indigent 
group (50-69 years old) when subjects without 
or with only slight ventilatoiy functional impair- 
ment were compared. Residual volume was cor- 
respondingly lower for the community residing 
group than for the indigent group when subjects 
in the sixth and eighth age decades were compared. 

It is therefore clear that inferences drawn about 
age changes in certain physiological characteristics 
may be biased when based on observations made on 
institutionalized subjects. 

Investigators in the Psychology Section are 
providing information on the psychological per- 
fomiance and personality characteristics on a 
group of subjects (aged 20-101) selected from up- 
per educational and socioeconomic levels. In ad- 
dition to standard tests of intellectual perform- 
ance, standard questionnaires to evaluate person- 
ality characteristics are being administered. Lab- 
oratory tests of learning ability under a variety of 
experimental conditions have been developed and 
applied to subjects over the age range of 20 to 101 
years. Estimates of reaction time, a-frequency of 
the electroencephalogram (EEG), spinal i-eflex 
amplitude, heart rate variability and motor time 
have been recorded under standardized condi- 
tions. 

In the psychophysiological progTam, a relation- 
ship has been found between age and brain-wave 
frequency of the electroencephalogram. The ob- 
served correlation of 0.59 (N = 55) shows that the 



period (the reciprocal of frequency) of an individ- 
ual's brain potentials is strongly associated with 
his age, and that chronological age accomits for 
35% of the variance in brain- wave frequency. 

A low positive correlation of 0.23 (N = 55) was 
obtained between age and mean reaction time, 
while a correlation of 0.71 between mean reaction 
time and mean brain-wave period of the electro- 
encephalogram was observed. The latter correla- 
tion is a significant and interesting finding. By 
means of a partial correlation analysis, the rela- 
tion between age and reaction time was determined 
under conditions where brain-wave frequency is 
held constant for all subjects. This partial correla- 
tion, which ])roved to be negative^ shows that 
brain-wave f I'equency can account for the increase 
observed in reaction time with age. Thus, an age- 
associated, central nervous system factor has been 
identified which may be the factor behind age- 
associated slowing in motor responses. 

Thus far, in the two verbal-learning experiments 
in which subjects (selected from upper educational 
and socioeconomic levels) participated the per- 
formance of the younger group was superior to 
the older (60 and above) and the slower the pace 
the better the performance. In addition, age dif- 
ferences were greatest at the fastest pace and 
smallest at the slowest pace. Results of the two 
verbal-leai'ning experiments with subjects of lower 
educational and socioeconomic levels were some- 
what equivocal. Further investigation is in 
progress. 

The extent to which judgments of stimuli are 
affected by preceding stimuli (context effects) have 
been implied as a possible source of age differences 
in recent perceptual studies. Using time esti- 
mation of visually perceived intervals to provide 
a measure of context effects, substantial context 
effects were produced but no age differences were 
found for this perceptual task. 

Preliminary cross-sectional analyses of the data 
collected on subjects from the upper educational 
and socioeconomic levels showed : ( 1) Four of the 
ten factor scores of a personality questionnaire 
(Guilford-Zimmennan Temperament Survey) 
were related to age. General activity, ascendance, 
and sociability scores decreased with age, and the 
restraint measure increased with age. (2) Of the 
four verbal fluencj' measures (Southern California 
Tests of Creative-Thinking Abilities), only Word 



NATIONAL HEART INSTITUTE 139 

Fluency (how rapidlywords containing a specified (r=— 0.44). (4) On a vocabulary measure, sub- 
letter are produced) was related to age (r= —0.21). jects above age 60 were somewhat superior to the 
(3) A measure of reproducing geometric designs group below 60. Almost every subject was above 
from memory (Benton Eevised Visual Retention the national average based on extensive adult 
Test) was most highlj"^ correlated with age norms. 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS 

DISEASES 



INTRODUCTION 

The direct research activities benefited this year 
by availability of additional space in Buildings 
5 and 8 and renovation of a number of laboratory 
areas. Next year will see additional space read- 
justment which will permit more adequate utiliza- 
tion of resources. The staff of the Laboratory of 
Biology of Viruses moved into new quarters on 
the third floor of Building 5, releasing additional 
space in Building 7 for expanded work in acute 
respiratory viral diseases. Additional space is 
also available for the Laboratory of Parasitic 
Diseases and Laboratory of Parasite Chemo- 
therapy in Building 5. The Laboratory of Tropi- 
cal Virology occupied enlarged laboratories 
renovated for both highly infectious agents and 
for ordinary virologic procedures. 

After twenty-five years of distinguished re- 
search in the Public Health Service, Dr. Harry 
Eagle retired to accept an academic position. The 
Laboratory of Cell Biology of which he was Chief 
was incorporated into the Laboratory of Biology 
of Viruses under Dr. Karl Habel as a Section. 
It remains essentially intact and continues produc- 
tive studies in cell biology. 

The Laboratory of Germfree Animal Research 
established a new Experimental Pathology Sec- 
tion under Dr. Edwin Lerner. This Section will 
support ongoing studies in germfree life and also 
provide histopathologic consultation to NIAID 
investigators. 

The Laboratory of Tropical Virology in which 
the Middle America Research Unit is administra- 
tively located secured expanded space in Bethesda, 
as well as improved facilities in the Laboratory 
in the Panama Canal Zone. Dr. Alexis Shelokov, 
Chief of the Laboratory, returned to Bethesda in 
July and relinquished his duties as Director, 
MARU to Dr. Henry K. Beye formerly with the 
Laboratory of Clinical Investigation. An Epi- 



demiology Section was established in MARU in 
addition to other previously established sections. 

On his return from a year of study in Europe, 
Dr. Carl L. Larson was assigned to the Office of 
the Director, NIAID, to conduct an evaluation of 
the total NIAID research program emphasizing 
particularly the extramural training program. 
Dr. Cornelius B. Philip succeeded Dr. Larson as 
Director of the Rocky Mountain Laboratory and 
Dr. Herbert Stoenner became Assistant Director. 
Dr. William Hadlow rejoined the staff of RML as 
Head of the Pathology Section. Plans were 
initiated for the construction of an animal house 
and insectary with new funds provided by the 
Congress. 

Dr. Don Eyles of the Laboratory of Parasite 
diemotherapy established a field station in Kuala 
Lumpur, Malaya on the grounds of the Medical 
Research Institute to investigate more intensively 
the problem of simian malaria. He is assisted by 
two NIAID staff members and local technical 
help. 

Although the position of Chief, Laboratory of 
Immunology has not been filled, research work of 
that laboratory is proceeding successfully under 
the able guidance of Dr. John Tobie, Acting Chief. 
One of the successful ventures of the Laboratory 
of Immunology which influences work throughout 
the NIH is the series of weekly seminars in immu- 
nologic subjects by staff members or outside 
scientists. A highlight was a special seminar in 
honor of the late Dr. Jules Freund, the first Chief 
of the Laboratory of Immunology, presented in 
December by Dr. Merrill Chase. 

As in the past, the Institute participated in the 
NIH Research Associate Program. Recent medi- 
cal graduates who can fulfill their military duty 
by a commission in the Public Health Service are 
selected and assigned to research laboratories. 
Seminars and lectures provide a scope of interest 
but the principal value is in actual research es- 

141 



I 



142 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



perience luider the guidance of a preceptor. This 
is the fifth year of the program. It is already 
clear that these yoimg medical scientists form an 
important reservoir of talent for the staif of this 
Institute and other research centers in universities 
and medical schools. 

As a part of the United States effort to establish 
better-scientific communication with the USSR, 
this Institute sponsored the U.S. Infectious Dis- 
ease and Microbiology Exchange Mission. A 
group of well known virologists, headed by Dr. 
Robert J. Huebner, Chief, Laboratory of Infec- 
tious Diseases, NIAID visited research institu- 
tions and laboratories in Russia for four weeks in 
the spring. Other members of the delegation 
were : Dr. Robert M. Chanock, Laboratory of In- 
fectious Diseases, NIAID; Dr. Fred M. Daven- 
port, University of Michigan; Dr. W. McD. 
Hammon, University of Pittsburgh; Dr. Edwin 
H. Lennette, California State Department of Pub- 
lic Health ; and Dr. Alexis Shelokov, Laboratory 
of Tropical "Virology, NIAID. As a further step 
in the international exchange of scientific infor- 
mation, this Institute was host to Dr. Nicolai 
Petrovich Yelinov, Deputy Director of the Lenin- 
grad Chemical-Pharmaceutical Institute, from 
September until December, who worked in the 
laboratoi'y of Dr. Chester Emmons, LID, investi- 
gating mycotic diseases. 

The Institute has continued to assign carefully 
selected staff scientists to other research labora- 
tories either to pursue their own line of investiga- 
tion or to learn new concepts and techniques. Dr. 
Eugene C. Weinbach, Laboratory of Parasitic 
Diseases worked at the Wenner-Grens Institute, 
University of Stockholm, Sweden. Dr. Philip 
McMaster, Laboratory of Immunology is at Pas- 
teur Institute, Paris. Dr. Maryjane K. Cook, 
Laboratory of Infectious Diseases continues her 
studies at Max- Planck Institut f iir Virusf orchung, 
Tubingen, Germany. Dr. Kelsey C. Milner, 
Roclfy Mountain Laboratory, began work at The 
Karolinska Institut, Stockholm, Sweden in Octo- 
ber. Dr. Sanford Stone started a year's work at 
the Hopital Broussais, Paris. Dr. J. Frederick 
Bell was awarded a Guggenheim Fellowship 
which provided him an opportunity to visit re- 
search laboratories in Middle Europe interested in 
tularemia. In December, Dr. William J. Jellison 
was assigned to the Pan American Sanitaiy Bu- 



reau to conduct investigations at the Zoonoses Cen- 
ter, Azul, Argentina. 

Once again the Institute was host to many scien- 
tific guest workers who remained from several 
days to several months. Five foreign scientists 
joined the Institute in the Visiting Scientist pro- 
gram to engage actively in curernt investigations. 
Six scientists supported by Fellowships worked 
with members of the Institute staff'. ■ 

Each year there is increasing demand and need f 
for training oportunities at the Institute. Ap- 
proximately 25 Visiting Scientists, Fellows, Re- 
search Associates, and Clinical Associates at the 
postdoctoral level engaged in research activities 
in a training capacity. It is clear that the various 
Institutes at NIH have the oppoilunity to play a 
major role in the postdoctoral degree education of 
biomedical scientists in tliis country. It is a role 
which this Institute can pursue eft'ectively, be- 
cause of its resoures and interest in a wide variety 
of research problems in infectious and allergic 
diseases. 

A long felt need in the study of viruses as a 
cause of disease is a readil}' available supply of 
uniform reference antisera and antigens. Virolo- 
gists find that with the increasing number of iso- 
lates (now numbering in the hundi-eds) their in- 
vestigations are curtailed by lack of identifying 
reagents. To fill this need, the Director, NIH re- 
quested this Institute to establish a Viral Reagent 
pi-ogram under guidance from selected scientists 
and representatives of interested Institute Coun- 
cils. This effort extends the support which the 
National Cancer Institute already has given for 
reagents of the enterovinises and this Institute for 
the adenoviruses. Panels of experts were estab- 
lished for enteroviruses and adenoviruses and will 
be established for the arthropod-borne viruses. 
The panels will write specifications for reagents 
and determine procedures for monitoring the 
quality of the material produced under contract. 

As new laboratory methods for uncovering 
viruses are developed, and their role as a cause of 
acute respiratory disease determined, it has be- 
come clear that vaccines for immunization will be 
needed as soon as possible. This Institute has ini- 
tiated a Vaccine Development Program which is 
intended to fill the gap between tlie fundamental 
research conducted in laboratories and the produc- 
tion of tested prophylactic vaccines suitable for 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



143 



use by practicing physicians. The rapid advance- 
ment of research accomplishments in respiratory 
diseases demands equal effort to translate these 
into practical usefulness. The Institute will be 
advised by a Board of outside scientists and staff 
members on specific developmental problems and 
the particular competence of various laboratories 
to fulfill the project requirements. 

The following pages describe the accomplish- 
ments of scientists working in Institute labora- 
tories. They comprise a wide spectrum of research 
endeavor. All projects have been significant and 
contributed in A-arious measure to scientific knowl- 
edge and to research techniques. Of special in- 
terest has been the development of procedures for 
utilizing inmate volunteers in the studies of minor 
respiratoiy illnesses and malaria. The excellent 
clinical facilities at the Clinical Center and the 
carefully controlled laboratory procedures insured 
a high degree of precise clinical measurement and 
safety. In a short time with relatively little cost 
the project has given valuable information which 
otherwise would have been impossible or inordi- 
nately time consuming and expensive. 

Advancements which are jDarticularly timely 
and influential are: the demonstration that the 
Eaton agent, an important cause of respiratory 
illnesses, is a pleuropneumonia-like-organism ; the 
description of the first cases of Venezuelan equine 
encephalitis in Central America; the axenic cul- 
ture of Entamoeba histolytica; demonstration by 
immunofluorescence of antibody production in hu- 
man malaria ; and the evidence for a new "foreign" 
cell antigen produced by polyoma virus as it trans- 
forms normal cells to tumor cells which is rejected 
by the innnunologically competent adult animal 
without tumor formation, whereas immunologi- 
cally tolerant suckling animals cannot reject the 
new antigen and tumors develop. Other equally 
important findings are included in the following 
report of activities. 



LABORATORY OF CLINICAL INVESTIGA- 
TION 

The program of the Laboratory has been en- 
larged by the development of projects in viral 
respiratory disease, immunology, mechanism of 
fever, and malaria. In contrast to previous years 



when optimum bed occupancy was achieved only 
with difficulty, the daily census has been main- 
tained near 100 percent by reason of the volunteer 
program, and there is now a waiting list for other 
cases which permits a more careful selection of 
those to be admitted. 

Viral Infection of Volunteers 

The largest project of the laboratory has been 
the study of viral respiratory disease in prisoner 
volunteers. During the year, the organization and 
function of this activity has become more efficient. 
A total of 189 volunteers have been inoculated 
with one of 14 different respiratory viruses or 
the Eaton agent. The principal purpose of these 
studies has been to determine the capacity of the 
agents to cause illness and to study the virological 
and immunologic resjionse to inoculation. The 
viruses tested were, with exception of influenza, 
suspected of causing significant amounts of re- 
spiratory viral infection, but their role had not 
been defined. Thus, a determination of their ca- 
pacity to cause illness represented an important 
step in assessing their role in the total picture of 
respiratory disease in the general population. 

A classical type of moderately severe viral in- 
fluenza has been produced in susceptible volunteers 
while a milder disease or no illness occurred in 
those with specific antibody. Illness and viral 
excretion began by the second day after inocula- 
tion and virus was still present in some cases 5 or 6 
days later, and after recovery from illness. These 
studies have demonstrated a type of infection and 
illness which lends itself to many kinds of clinical 
study. 

Despite many close biologic and biochemical 
similarities it -was found that Coxsackie A-21 (the 
Coe agent) caused an acute, febrile flu-like illness 
while Coxsackie A-24 (the Pett virus) caused no 
illness. Virus was repeatedly isolated from the 
throat of volunteers given A-21, while A-24: was 
fomad only in rectal swabs. Several higher-type 
adenoviruses have been shown to produce disease 
when inoculated by cotton swab into the conjunc- 
tiva beneath the lower lid but not when given in- 
tranasally or by spray into the throat. In all 
situations, however, viral infection occurred with 
both throat and anal swabs positive. The higher 
type agents appear to be less virulent than lower 



144 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



type adenoviruses, as judged by comparison with 
earlier studies. 

Keo-viruses obtained from human sources failed 
to produce illness although viral infection oc- 
curred in susceptibles. Recently, a common cold 
syndrome with rhinorrhea, malaise, and in some 
cases, fever, has been produced by three new and 
presently unclassified enteroviruses. The incuba- 
tion period was only a few hours. 

Salmonella Carriers 

Studies of 26 chronic salmonella carriers re- 
vealed that gallstones present in each case con- 
tained salmonella (most were S. typhosa) . When 
these stones were incubated in high concentrations 
of antibiotics, salmonella persisted and grew in 
subsequent cultures not containing antibiotics. 
Consistent with these findings was the observation 
that antibiotic treatment alone was not effective 
in curing the carrier state. On a basis not yet 
fully explained, was the further observation that 
cholecystectomy alone was often not effective in 
curing carriers, suggesting that infection in sites 
other than gallstones perpetuate the carrier state. 
A few instances of positive cultures from liver 
tissue were found. Finally, it was found that 
lasting cure occurred if antibiotic treatment was 
combined with cholecystectomy. It was estimated 
that over 300 cases and 3 deaths from typhoid 
fever occurred in contacts of this group of patients. 



antibody appearing approximately two weeks 
after onset of illness and reaching a peak about 
one month later. Titers as high as 1/5120 were 
frequently reached. Following recovery the titers 
slowly decreased. In connection with these stud- 
ies, it was found that the concentration of circu- 
lating gamma globulin in the malaria patients 
increased during infection, and the highest values 
of gamma globulin coincided closely in time with 
the occurrence of the highest titer of malaria 
antibody. 



4 



Mechanisms of Fever 

A new program this year has been the investiga- 
tion of host response to fever, and the causes of 
fever and related phenomena in certain febrile 
diseases of obscure origin. The studies are pres- 
ently centered around the disease known as 
familial Mediterranean periodic fever or familial 
polyserositis. It is an inherited disease of certain 
persons of Jewish, Armenian and sometimes of 
Turkish descent. It is characterized by many 
years of progressive, disabling attacks of non- 
infectious fever and peritonitis unrelieved by any 
presently known treatment. Studies here have 
been concerned with the effect of the recurrent 
fever on mechanisms of heat loss when the patients 
are stressed in a low temperature chamber. It 
appears now that patients with polyserositis have 
lost some responsiveness to cold stimulation as 
compared with normals. 



Malaria in Volunteers 

Following demonstration of human infection 
with the simian vivax-type malaria, Plasmodium 
cynomolgi iastianeUii, several prisoner volunteers 
infected with this agent, and others inoculated 
with human vivax strains were studied at the 
Clinical Center. In addition to the usual para- 
sitologic determinations, radioactive chromium 
survival tests, of erythrocytes excretion of adrenal 
steroid metabolites, liver function, a number of 
other parameters were measured. Among other 
findings was the observation that a significant re- 
duction in serum haptoglobin concentration took 
place in association with hemolysis of the red blood 
cells. 

The most significant observation, however, was 
the demonstration of fluorescent stainable malaria 



Fungus Disease 

The program in systemic fungus infection has 
continued in a vigorous way. A number of 
clinical reports have been made of treatment of 
various kinds of fungus disease with ampliotericin 
and the new agent of Hoffman-La Roche, X5079C. 
Both drugs are extremely useful in treatment of 
fungus disease as described in the following table : 



Blastomycosis 

Histoplasmosis 

Sporotrichosis 

Cryptococcosis 

Coccidioidomycosis 
Candidiasis 



Amphotericin 



Good 

Good 

Good 

Good 

Slight 

Moderate,. 



X5079C 



Good. 

Good. 

Good. 

Doubtful. 

Doubtful. 

Doubtful. 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



145 



Since amphotericin causes significant renal tox- 
icity and local irritation, especially when intro- 
duced into the spinal canal these features of treat- 
ment have been studied. It was found that a per- 
sistent renal tubular lesion characterized by 
prolonged depression of para-amino hippurate 
clearance is caused by amphotericin. Further- 
more, recent studies of biopsies of kidney from 
patients under treatment reveal necrosis of 
glomeruli. This is an unusual renal lesion and is 
under detailed investigation in dogs. So far no 
effective means has been found to control the irrita- 
tive effects of the drug on the central nervous 
system after intrathecal instillation. The nerve 
damage encountered has been so severe that local 
use of the agent in meningitis (cryptococcal) will 
henceforth be administered only to those cases in 
which it is considered an absolute essential. 

The compound X5079C was found to cause a 
prompt rise in bromsulf alein retention despite only 
slight evidence of interference in other kinds of 
liver function. This received careful examination 
in dogs and the effect is now revealed to be a com- 
petition of the antibiotic for the excretory route 
for bromsulfalein. The exact mechanism has not 
been determined. Prolonged treatment with the 
agent causes some liver damage but it appears to 
be sufficiently slight to permit therapeutic use of 
the drug. 

Penicillin Derivatives 

Two new penicillin derivatives have been tested 
in staphylococcal infection. The agents, Pros- 
taphlin, manufactured by Bristol Laboratories, 
and SKF JN'o. 12141, manufactured by Smith, 
Kline & French, are similar in that they are sub- 
stantially resistant to destruction by penicillinase, 
they are significantly more active on a weight basis 
against sensitive staphylococci than dimethoxy- 
phenylpenicillin, the first of the penicillinase re- 
sistant compounds (see last year's report), and 
they are active after oral administration. Limited 
clinical studies have revealed that these agents are 
effective in the treatment of staphylococcal infec- 
tion, including those resistant to older forms of 
penicillin. Biochemical studies on these com- 
pounds have also been made and will be described 
elsewhere in the report. 



Penicillin-Resistant Staphylococcal Infections 

Work in the past three years on the problem of 
penicillin-resistant staphylococcal infections has 
developed into two distinct problems. The first 
phase of the investigation dealt with the nature 
of the resistance of staphylococci to penicillin. It 
was established that naturally-occurring patho- 
genic strains of Staphylococcim aureus which are 
resistant to penicillin, are resistant by virtue of 
their containing an enzyme, penicillinase, which 
rapidly destroys penicillin G. A series of papers 
have been published on the biochemical properties 
of this enzyme as it occurs in S. aureus and on its 
role in penicillin resistance. 

The second phase of the investigation has been 
concerned with the many new penicillins that have 
become available in the past two years as the re- 
sult of a technologic development in the field of 
penicillin chemistry; namely, the ready availa- 
bility of 6-aminopenicillanic acid as the immediate 
precursor of a wide variety of penicillins previ- 
ously unknown. When it was determined that re- 
sistance of staphylococci was based on their ability 
to destroy penicillin G at a rapid rate, it became 
evident that a possible solution to the problem 
would be to find a modification of penicillin which 
would be resistant to the destructive action of 
penicillinase and yet retain antibiotic activity. 
Wlien 2,6-dimethoxyph6nylpenicillin was given 
to the Laboratory for clinical trial, it was very 
soon found that this new penicillin met the essen- 
tial biochemical criteria and offered therapeutic 
promise. Our laboratory, by virtue of being the 
first to obtain staphylococcal penicillinase in large 
amounts in concentrated and purified form, was 
able to establish the absolute rate of destruction of 
the new penicillin by staphylococcal penicillinase 
and show it to be more resistant than penicillin G 
by a factor of about 200. The consistently good 
therapeutic results obtained with this drug on ex- 
tensive clinical usage in the past year have tended 
to verify the conclusions drawn from the labora- 
tory findings. 

Similar techniques have enabled us to evaluate 
all the new penicillins which have been offered to 
us for clinical trial including an isoxazolyl peni- 
cillin which is not only resistant to penicillinase 
but is also sufficiently stable to acid to pemiit its 



146 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



oral use. Such a penicillin would have an advan- 
tage over 2, 6-dimethoxyphenylpenicillin which is 
not effective after oral administration and must 
be given intramviscularly or intravenously. 

Clinical Immunology 

Association with the Clinical Immunology Sec- 
tion of the Laboratory of Immunology has pro- 
vided the Laboratory of Clinical Investigation 
with an increasing number of laboratory tech- 
niques to support clinical studies. The Immuno- 
electrophoresis technique, agar gel diffusion meth- 
ods, and fluorescent antibody techniques have been 
used to study the serum of patients with the 
nephrotic syndrome, lupsu erythematosus, and 
other disease entities under study by members of 
the laboratory. A group of 60 patients with the 
nephrotic syndrome studied over the past eight 
years are being followed to evaluate the effect of 
steroid therapy. Similar studies are being made 
with a group of patients with lupus erythematosus 
and in addition, these patients are being evaluated 
for the degree of skin test hypersensitivity to 
autologous leukocytes. 

Mouse Plasma Cell Tumors 

Two years ago it was found that mice injected 
intraperitoneally with Freund's adjuvant and cer- 
tain antigens developed chronic ascites containing 
a high titer of antibody to the administered anti- 
gen. Later it was shown that a certain number 
of animals, which were strain-related, developed 
rapidly fatal plasma cell tumors. Further, it was 
observed that the tumor-bearing mice were poor 
producers of antibody. These tumors have been 
found to produce predominantly one or another 
types of globulins (alpha, beta, gamma) and cur- 
rently studies are under way to measure the rela- 
tive responsiveness to antigenic stimulation of 
mice with these tumors. 

LABORATORY OF IMMUNOLOGY 

Passive Transfer of Allergic Encephalomyelitis 

The passive transfer of allergic encephalomy- 
elitis was accomplished when lymph node cells 
from sensitized Strain 13 inbred guinea pigs were 
transferred to normal Strain 13 recipients. Fur- 



thermore, tliis exprimental autoimmime disease 
was transferred from sensitized adult Strain 13 
guinea pigs to newborn Strain 13 animals. Strain , 
2 inbred guinea pigs are relatively resistant to al- 
lergic encephalomyelitis and the disease is not 
transferred to the Fl generation animals of a 
Strain 2-13 cross. The Fl animals evidently in- 
herit the Strain 2 resistance. Passive transfer 
with viable lympoid cells represents an important 
tool in the study of imniune plienomena. In histo- 
compatible animals, the variable of transplanta- 
tion immunity is eliminated, the transferred cells 
thrive, and tlie cells go on to immune maturity in 
the recipient. 



fl 



Antibody Production and Gamma Globulin in 
Human Malaria 

For the first time, the course of antibody pro- 
duction has been followed in human malaria. It 
has been possible not only to detect but to titrate 
antibody by the fluorescent antibody technique 
and to correlate this with the appearance of the 
parasites in the peripheral circulation. In 4 spo- 
rozoite-induced Plasmodian vivax infections in 
human volunteers antibody was detectable several 
days after the parasites appeared. In a typical 
case, antibody rapidly rose to a seinim dilution 
level of 1 : 2,560, was maintained at this level for 
about 40 days after which is gi'adually fell and 
remained at a low level for the period of observa- 
tion (121 days). In 5 patients infected with the 
B strain of P. cynomolgi essentially the same re- 
sults were obtained and certain of the patients 
still had detectable antibody 1 year after infection. 
The patients showed gamma globulin levels above 
the normal limits. The maximun^ ganuna globul in 
rise tended to occur at the same time as the maxi- 
mum antibody titer with a median difference of 3 
days. The ability to follow antibody production 
and increases in ganuna globulin levels in con- 
trolled malaria infections provides some very basic 
information necessary in the program of world- 
wide eradication of malaria. 



Proteolytic Enzjnme of Human Spleen Purified 

A method has been established for the isolation 
and purification of a proteolytic enzyme from hu 
man spleen which cuts human albumin into large 






NATIONAL INSTITUTE OF ALLERGY AND INTECTIOUS DISEASES 



147 



precipitating fragments. By column cliromatog- 
raphy tlie enzyme has been shown to be a single 
substance. Its electrophoretic mobility, pH activ- 
ity curve and catlieptic activity has been deter- 
mined. Four large fragments of human albmnin 
resulting from the degradation of the native pro- 
tein by the human enzyme have been detected by 
Immunoelectrophoresis. This study combines sev- 
eral fimdamental problems bearing on the nature 
of antigenicity and the possible roles of enzymes 
in the degradation of antigens as a primary step 
ia antibody formation. 

Lupus Serum Reacts With Mammalian Chromo- 
somes 

Fluorescent antibody studies have demonstrated 
that sera from certain patients having lupus 
erythematosus •when applied to mammalian 
chromosome preparations, possessed anti-nuclear 
factors. In the case of human chromosomal prep- 
arations all 46 chromosomes reacted and became 
fluorescent. '\Vlien sera from three patients with 
Sjogrens syndrome were utilized, the chromosomes 
did not become fluorescent even though the sera 
had anti-nuclear factors. It may be possible to 
find sera or fractions of sera which are more spe- 
cific in their chromosomal reactions. The anti- 
nuclear factors found in the sera of patients with 
the so-called autoinunune diseases are of import- 
ance for a better imderstanding of the mechanisms 
involved. 



Genetic Control of Ganuna Globulin Allotypes 

In studies on the genetic control of serum allo- 
types in rabbits, it was previously shown that 
RGG-I and EGG-II allotypic determinants are 
under the genetic control of an allelic pair of 
autosomal genes. Further studies have revealed 
three other gamma globulin allotypic determinants 
which are under the genetic control of a three 
allelic system at a second gene locus. Further- 
more, two allotypic determinants were either both 
present or absent from all sera tested indicating 
genetic control by a single allele or two closely 
linked alleles. In addition a gamma globulin at a 
third locus has been found. The distribution of 
allotypes varied considerably among several rabbit 
colonies. The selective breeding of rabbits is in 

643351—62 11 



progress to establish genetically defined lines of 
rabbits based on the known gamma globulin al- 
lotypes. 

Allergic Thyroiditis 

Delayed hypersensitivity to thyroid extract has 
been successfully transferred within Strain 13 
inbred guinea pigs. Intact lymph node cells from 
animals actively inunimized to thyroid extract 
were injected into normal recipients. Successful 
transfer was accomplished only if the lymph node 
cells were taken early after innnunization of the 
donors. Cells taken 5 days after sensitization suc- 
cessfully transferred skin test hypersensitivity. 
Lesions of the thyroid were produced by the trans- 
fer of lymph node cells in about 30 percent of the 
recipients. 

LABORATORY OF BACTERIAL DISEASES 

The research program in the Laboratory of 
Bacterial Diseases has continued to develop in the 
same general areas noted in last year's report. The 
major emphasis has been on the projects on In- 
tracellular Parasitism and Stapliylococcus. 

In Vitro Cell Antibody 

In the course of the project on Intracellular Par- 
asitism a study was midertaken of the production 
of specific antibody by mammalian cells in vitro. 
This study has been reasonably rewarding and 
offers considerable promise. Accordingly, at pres- 
ent, the major effort on this project is devoted to 
this line of investigation. 

Specifically, cells are obtained from the peri- 
toneal cavity of laboratory animals, usually the 
guinea pig. A method has been developed which 
yields a high proportion of mononuclear cells. 
Further separation of the ceU types results in ex- 
plants which are composed almost entirely of this 
type of cell. Cells removed from immune animals 
and properly handled and cultured elaborate spe- 
cific antibody in vitro for days or even several 
weeks. The explants result in growth of sheets of 
cells which are derived from the mononuclear cells. 
On glass they become stellate cells but retain some 
of the characteristics of macrophages. Such tissue 
cultures can be maintained for long periods and be 



148 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



carried through at least a number of subcultures. 
The antigen usually has been egg albmnen ; the test 
procedure, hemagglutination of specifically sen- 
sitized red blood cells. Wliile production of anti- 
body in vitro has been sporadic, significant titers 
have been obtained, and in a number of experi- 
ments antibody has been detected for periods of 
several weeks. In comparison with initial cell 
homogenates and with supernates from cells main- 
tained under non-optimum conditions, the amount 
of antibody produced exceeds that which can be 
accounted for by release of preformed antibody 
and certainly represents new protein synthesis 
after removal of the cells from the animal. There 
is still a question as to the type of cell which is 
producing antibody and also whether these par- 
ticular cells have undergone multiplication even 
though general growth of the tissue culture has 
occurred. This system allows further study of the 
factors involved in continued antibody production. 

Staphylococcus Virulence 

In the project on Staphylococcus there is con- 
tinued emphasis on the factors responsible for 
virulence of the organism and on basic studies 
of methods of identification of pathogenic strains 
of the organism. Gel-diffusion immune precipita- 
tion techniques have been applied to standard ref- 
erence strains of Staphylococcus. Employing 
methods which tend to eliminate minor and non- 
specific cross reactions the antigenic mosaic of such 
strains has been characterized. This technique 
seems promising for the development of a system 
of identification. If reference spectra consisting 
of precipitation patterns formed with specific ref- 
erence antigens and antisera can be established for 
staphylococci, this would form a solid basis for 
the identification of strains. 



Brucellosis Diagnosis 

We continue to collaborate with other Brucella 
research centers throughout the world in the de- 
velopment and standardization of laboratory tests 
for species identification, and for diagnosis of Bru- 
cella infection. Brucellosis is still a disease of 
world wide importance in relation to the welfare 
of mankind. Control of the disease in domestic 
animals rests in part upon regional epidemiologi- 



cal studies which require precise identification and 
classification of strains. A unified effort is cur- 
rently undei- way in an attempt to clarify Brucella 
taxonomy. This undertaking is mider tlie aegis of 
the Subcommittee on Taxonomy of the Genus 
Brucella of the International Conmiittee on Bac- 
teriological nomenclature, of wliich the Chief, Lab- 
oratory of Bacterial Diseases, is a member. 

LABORATORY OF BIOLOGY OF VIRUSES 

Two major changes in the Laboratory' of Biolog)' 
of Viruses during 1961 have resulted in an expan- 
sion of its activities. Between July and Septem- j j| 
ber, all units of our original laboratory were moved ' 
from their scattered locations in Building 7 to be 
together on one floor in Building 5. The second 
change was the transfer as a section to the Labo- 
ratory of Biology of Viruses of the remainder of 
the Laboratory of Cell Biology after the retire- 
ment of Dr. Harry Eagle. 



Virus-Host Relationship 

Investigations of relationships between the virus 
and its host cell have continued to be the greatest 
effort of the laboratory. Four different units are 
working in this important general area, attempt- 
ing to define the biochemical events, their sequence, 
site and inter-relationships. Tliese studies repre- 
sent both basic and practically oriented research. 
The basic and more direct goal is to determine the 
mechanism of protein and nucleic acid syntheses 
in the normal cell, the virus being used only as a 
self-replicating identifiable biochemical macro- 
molecule. The other more remote goal is the de- 
velopment of knowledge concerning virus invasion 
and multiplication which might give a lead as to 
a logical point of attack in chemotlierapy of virus 
diseases. Studies on poliovirus in HeLa cells have 
shown that viral protein and viral UNA syntheses 
are closely coordinated in time and increase only 
a short time before whole infections virus can be 
demonstrated. By the use of inhibitors there is 
evidence that the synthesis of viral RNA may be 
at least partially dependent ujDon protein synthe- 
sis. Viral protein antigens appear in both the 
nucleus and cytoplasm, but evidence is lacking as 
to whether this is the result of synthesis in both 
areas of migration after synthesis. Studies on 



41 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



149 



vaccinia virus — a DNA virus — suggest that tlie 
synthesis of viral DNA and viral protein are dis- 
sociated. Work with Coxsackie virus — a KNA 
virus — shows precursor viral ENA present in the 
nucleus early after infection, declining by 3 hours 
and again increasing at 6 hours. Viral E.NA in 
the cytoplasm decreases until 2 hours, then rises 
continuously to eventual high levels. 

Virus Structure 

Three units have carried out studies on chemical 
and physical structure of virus as such. The pro- 
tein coat of poliovirus has been isolated and found 
to contain no free N-temiinal amino acid residues. 
The subunit of this protein appears to have a 
higher molecular weight than the predicted 20,000. 
A base analogue — 5 fluorouracil — has been suc- 
cessfully incorporated into the EISTA of poliovirus 
to the extent of replacing 30% of the normal ura- 
cil, but with retention of biological properties of 
the virus. This "abnormal" virus continues to 
have the properties of the parent normal virus in 
regard to infectivity and host range susceptibility. 
Coxsackie virus has been shown by electron micro- 
graphs of purified virus to have an outer envelope 
and a dense inner core. 

Virus Inhibitors 

An important practical contribution to virus 
research technology has been made by the group 
studying virus inhibitors. The previously dem- 
onstrated inhibitor of plaque formation by certain 
genotypes of certain viruses has been identified 
as a sulfated polygalactone. Two different practi- 
cal methods for its removal from agar have been 
developed as well as materials found which can 
neutralize the inhibitor. This has made it possible 
to obtain plaques with strains of ECHO viruses, 
myxoviruses, arbor viruses, adenoviruses and ra- 
bies virus, all of which produced no plaques by 
standard methods. 



new "foreign" cell antigen appears. This "for- 
eign" antigen is rejected by the immimologically 
competent adult animal and tumor development 
is suppressed, whereas the suckling animal being 
immunologically tolerant cannot reject the new 
antigen and tumors develop. This demonstrates 
two important phenomena: 1) the interaction of 
a virus with a mammalian cell can induce a ge- 
netically stable change in the antigenic compo- 
nents of the cell, and 2) immmiological competence 
may be the limiting factor in the progressive de- 
velopment of a tumor after transformation has 
occurred. 

Melanin Granules and Mitochondria 

Important contributions have been made in the 
general area of normal cell metabolism unrelated 
to virus infection. In the application of the find- 
ing made last year that tetracycline antibiotics 
localized specifically on mitochondria and can be 
demonstrated by fluorescent microscopy, it has 
been found that melanin granules in melanoma 
tumors act like mitochondria thus adding another 
bit of evidence for the mitochondrial nature of 
these granules. 

The development of a mutant line of HeLa cells 
resistant to the toxic effects of a glucose analogue 
has led to the demonstration that this drug resist- 
ance is due to the presence of an inhibitor of 
hexose phosphorylation. This inhibitor may be 
related to akaline phosphatase since the latter is 
8 to 10-fold higher in the resistant cells. Studies 
using a DNA inhibitor in HeLa cells have pro- 
duced evidence that synthesis of protein and ENA 
in the nucleus is DNA-dependent, whereas their 
synthesis in the cytoplasm is not. In a continua- 
tion of studies on inborn metabolic defects, 
initiated by another investigator, current studies 
of cell cultures from gout patients show these cells 
to produce and release large quantities of purines 
compared to normal cells. 



. "Foreign" Cell Antigen 

A study of the mechanism of oncogenesis by 
polyoma virus has developed evidence that when 
the virus transforms normal cells to tumor cells it 
changes the genome of the cell in such a way that a 



LABORATORY OF PARASITE 
CHEMOTHERAPY 

The United States is an active participant in 
world-wide malaria eradication. Our principal 
contributions to the effort arise from basic and 



150 



ANlSrCTAL REVIEW OF INTRAMURAL RESEARCH 



clinical studies on the disease. In addition, we 
are committed to the same kind of investigations 
involving parasitic infections in general, with 
special emphasis on schistosomiasis and intestinal 
parasites. Because of the zoonotic character of 
simian malaria, as demonstrated here in 1960, 
and a large interest by the senior staff in overall 
problems of malaria, the main research effort dur- 
ing the year has been in that field. 

Malaria — Simian 

Man-mosquito-man transmission was accom- 
plished consistently with two different strains 
(B & M) of Plasrwodiwn Gynomolgi, even with low 
gametocytemias. Man-mosquito-monkey trans- 
mission was accomplished with each strain also. 

Infections produced by the inoculation of blood 
parasitized with the B or the M strain of P. 
cynomolgi exhibited no significant differences in 
clinical manifestations. However, when these 
infections were induced in volunteers by mosquito 
bites, several differences were noted; in the M 
strain, the duration and height of the fevers were 
greater, the tertian patterns were more numerous 
and typical, and splenomegaly more frequent. 

Malaria Studies in Malaya 

In Malaya, approximately 33 percent of pig- 
tailed macaques {Macaca nemestrina) and about 
40 percent of the long-tailed macaques {Macaca 
irus) are infected with a malaria. A new species 
of malaria was isolated from this latter species and 
was designated PlasTrwdiwm fieldi in honor of Dr. 
John Field. M. inis also harbored P. knowlesi and 
P. inwi. Hepatocystis semnopetheci was dis- 
covered for the first time in M. nerrhestrina. 

Malaria parasites were also demonstrated in 
Macaca irus and M. nemestrina leonina from Thai- 
land and from the latter monkey from East 
Pakistan. Plasmodiwm, hnoiolesi and P. inui, 
along with a species of Hepatocystis, were found in 
M. irus lacta from an isolated island off the east 
coast of Malaya. 

A significant proportion of all three Malayan 
species of leaf monkeys (Presiytis cristatus, P. 
oiscurus, and P. malalophos) were shown to be 
infected with malaria. Exact species determina- 
tion of the parasites have not been made. 



Anopheles liacheri was shown to be a natural 
vector of P. knowlesi and at least two, possibly 
three, tertian-type parasites so far imidentified. 
A. maculatus, A. simdiacus, A. leucosphyrus, A. 
umbrosu-s and A. letifer were found to bite simians 
but so far there is no evidence that any transmit 
malaria. Experimentally, it was shown that ^1. 
inacidutus, A. sundiacus, A. phillppinensia, A. 
hoclii and A, hacJceri are susceptible to infection 
with P. cynomolgi. 



Malaria — ^Human 



4 



Plasmodiwn-b falciparum from Colombia, South 
America was found to be resistant to several mem- 
bers of the important 4-aminoquinoline group of 
drugs, i.e., chloroquine, amodiaquine, and hydroxy- 
chloroquine. Because of the wide-spread use of 
these drugs, such resistance, if prevalent, could 
be of major significance. Resistance to mepaci'ine 
was present also. Resistance has now been shown, 
by certain malaria parasites, to each of the impor- 
tant groups of synthetic antimalarial drugs de- 
veloped in the last 30 years. 

A drug (BW-377C54), with reported activity 
against human malaria similar to that of cliloro- 
quine, failed to cure P. falcipaniin malaria. Also, 
the Colombia strain of P. falciparam appears to 
be resistant to this compomid. 

A study of the life-pattern of Venezuelan vivax 
malaria indicates that it has an intermediate-type 
latent period following treatment of the initial 
attack. This differs from the short- tenn (Ches- 
son) or long-term (St. Elizabeth) latent period of 
other strains. 

The chloroquine-resistant strain of P. falci- 
parum contains a characteristic component wMch 
can be detected m hydrolyzed ENA. This sug- 
gests a chemical method for identifying drug re- 
sistant strains of malaria. 

Biochemical Studies 

Analysis of ribonucleic acid derived from Plas- 
modiimi gallinacewn, P. ierghei, P. cynomolgi, P. 
inui and P. gonderi revealed the presence of nu- 
cleotides of the usual purine and pyrimidine bases : 
adenine, guanine, cytosine and uracil with adenine 
most abundant and cystosine the least abundant. 
In addition, nucleotides of hy|)0xanthine are found 
indicating the activity of adenosine deaminase. 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



151 



Significant binding of chloroquine to a compo- 
nent or components in plasma, but not to pure 
seinma albumin, was demonstrated. The degree 
of binding of this drug to blood cellular elements 
in vitro could be altered by a niunber of physical 
means. Antimalarial drugs, energy sources, and 
metabolic inhibitors were studied to determine 
their effect on the incorporation of amino acids 
into malaria protein. None of the antimalarial 
drags had an effect on this process or on glycolysis. 

Nucleic acid extracted from a pyrimethamine- 
resistant strain of P. gallinaceum hastened the de- 
velopment of resistance in a normal strain exposed 
to both drug and the extract. A decrease in chloro- 
quine sensitivity of P. ierghei has been observed 
foUowmg exposure of infected donor mice to 1,000 
roentgens of X-radiation. 

Nutrition and Malaria 

Nutritional deficiencies that favor the develop- 
ment of peak parasitemia in mice infected with 
Plastnodium ierghei include folic acid, panto- 
thenic acid, and niacin. 

Deficiencies suppressing parasitemia include 
para-aminobenzoic acid and riboflavin. Aininop- 
terin, a folic acid antagonist, also suppressed 
peak parasitemia. Thiamine deficiency appeared 
to have no effect. Deficiencies were studied both 
in the absence and presence of suppressive levels 
of chloroquine. In only one case did deficiencies 
have a reverse effect; pyrodoxine deficiencies ap- 
peared to favor the parasite in the presence of 
the drug but suppressed the parasites in the ab- 
sence of the drug. 

Intestinal Parasites 

Bephenium chloride, given for three or five 
days, was highly effective against hookworm in- 
fections and also removed 68 and 88 percent, re- 
spectively, of Trichuris tricMura worms. A rural 
population heavily infected with Ascaris lost 90 
percent of their worms when given single doses 
of piperazine. Hookworm and T. tincTiiura infec- 
tions were shown to persist for at least seven years 
in institutionalized mental patients. 

Tapeworms {Hymenolepis diminuta) exposed 
to mepacrine (Atabrine) in vivo were resistant to 
the action of the drug in vitro. This is interesting 



to follow up because it suggests a type of resist- 
ance not based on selection but upon physiologic 
adaptation of individual organisms. 

Evidence suggests that three anthelmintics, be- 
phenium, mepacrine, and diclilorophen, are ef- 
fective by inhibiting the enzymes located on the 
outer surface of tapeworms. 

Insect Tissue Cultures 

Lepidopteran tissues have undergone growth or 
extended maintenance in culture. In cultures of 
caterpillar hemocytes, St. Louis encephalitis 
(SLE) virus has been maintained up to 10 days. 

Prepupae and pupae fat body cells readily pro- 
liferate in hanging drop culture for 10-14 days ; 
SLE virus persists in such cultures and may in- 
crease between the 6th and 8th day. 

Virus-Mosquito Larvae Associations 

Larvae of Aedes aegypti and CuLex pipiens mos- 
quitoes acquired SLE viruses from an experimen- 
tally contaminated aquatic environment. The vi- 
rus persisted through the subsequent pupae stage 
and into the adult insect. There have been nine 
confirmed transmissions of the virus by bites of the 
Aedes and nineteen by the Culsx mosquitoes. 

Virus-Parasite Combinations 

Plasmodium galliTiaceum, reduced the quantity 
of circulatmg encephalomyocarditis virus in 
chicks. Additional experiments have continued 
to verify the role of P. ierghei in the transport 
of SLE virus to the central nervous system in mice 
when the virus is given intraperitoneally. 



LABORATORY OF PARASITIC DISEASES 

Axenic Culture of Entamoeba Histolytica 

Work on the cultivation of Entamoeba histoly- 
tica in the absence of any other living organisms 
has progressed steadily. Most important is the 
demonstration that the teclinique of developing 
such axenic cultures is applicable to more than 
one strain. A strain of the amoeba very recently 
isolated from a human patient has been established 
in axenic culture. Secondly, the culture medium 



152 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



has been further simplified, with the elimination 
of chick embryo extract. Also, it has been pos- 
sible to eliminate agar from the liquid overlay, 
a technical factor of importance in that it will 
eventually aid in evaluating culture growth. 

Freezing Protozoa 

Freezing of protozoa in liquid nitrogen has been 
developed to the point of practicability. Enta- 
moeba histolytica has been kept viable for at least 
10 months and Trichomonas vaginalis for six 
months ; there have been no losses in viability by 
the use of the technique. Other tests underway 
clearly demonstrate that Entamoeba invadens, 
Trichomonas hominis, T. gaUinae, and T. foetus 
can be maintained viable in liquid nitrogen. 
These experiences indicate that indefinite storage 
of protozoal strains is feasible. 

Filariid Larvae in Vilro 

The development of techniques for freeing 
microfilariae completely from blood cells and the 
demonstration of development of microfilariae to 
the sausage stage are significant advances. This 
is the first time that sausage stages have been 
found with any regularity in in vitro preparations. 
Additional work can now be expected to result in 
further development of filariid larvae in vitro. 
Survivial of adult Dirofilarla uniformis in chemi- 
cally defined tissue culture nutrients (NCTC 
109 -MO % serum) has been obtained for as long 
as 35 days. Microfilariae were discharged from 
the females during the first 2 to 3 weeks, only when 
serum was present in the medium. Under these 
conditions for maintenance of the filariids, glu- 
cose was transformed quantitatively to lactate. 

Nematode Cycle in Germfree Animals 

In the study of the development of Nematospi- 
roides dubius in germfree animals, the nematode 
has now been carried through its entire life cycle 
three times in the absence of bacteria. Larvae 
from the feces of germfree mice have been culti- 
vated axenically to the infective stage and then 
used to reinfect new germfree mice. It is note- 
worthy that infections thus produced in germfree 
animals are not as extensive as in conventional 



1 

an I 



animals; worm burden, worn size, and egg-pro 
duction appear reduced. This may represent an 
important lead in determining the factors respon 
sible for alterations in host-parasite balance. The 
possibility of a steroid action, which is in some 
way dependent upon the microbial activity in the 
intestinal tract, is suggested by the differences 
between conventional and germfree animals in 
respect to the worm recovery in male versus fe- 
male mice. 

Nutrition and Schistosomiasis 

The study on the relation of nutrition to schisto- 
somiasis in Puerto Eico is almost complete. Liver 
function tests returned to normal in all scliisto- 
some-infected patients who were placed on the 
high protein, high caloric diet. Stibophen (Fua- 
din) levels were higlier and maintained longer in 
the same patients. 

Studies on the mechanisms wliich produce 
higher blood levels of stibophen in Iiuman beings 
on high protein high calorie diet have been ac- 
complished in mice on complete semi-synthetic 
diets in which enhanced activity of stibophen is 
observed. The higher levels of drug are attribu- 
table to the acid-base relations of salts in the diet. 
With acidic salts Fuadin activity is enhanced. 
Similarly the efficacy of antimony potassium tar- 
trate, antimony (III) sodium meso-2 3-dimer- 
captosuccinate (Astiban) and pararosaniline pa- 
moate for killing mature Schistosoma mansoni in 
mice has been found to be increased up to 16 X in 
mice maintained on the semi-synthetic diet com- 
pared to mice on the commercial pellet diet. 

Experimental Schistosomiasis 

Continued work on the pathology of schistoso- 
miasis has revealed differences in response among 
mice, hamsters, and multimanmnate rats {Masto- 
mys) as to the vascular lesions seen in the liver. 
Mice and Mastomys develop more lesions than 
hamsters. The explanation for this may be that 
the lesions are at least ijartly due to allergic re- 
actions and the hamster is notably less reactive 
in this respect. The development of hepato- 
splenic schistosomiasis occurs in mice even with 
very small worm burdens; a longer period of time 
is required for the syndrome to appear. The cor 



! 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



153 



pulmonale picture produced in schistosome- 
infected mice by partial portal vein ligation pro- 
vides another experimental model for studying 
clinical scMstosomiasis. 



Biochemical Studies 

Investigations on Trypanosoina crusi have re- 
vealed the fixation of carbon dioxide by this organ- 
ism, with the incorporation of the CO2 into suc- 
cinic acid. Degradation of succinic and acetic 
acids from incubates containing C" labeled glu- 
cose has established that most of the carbons of 
these acids are derived from glucose. Preliminary 
fractionation of the phospholipids of T. eruzi has 
yielded serine and ethanolamine as well as an un- 
identified niiJiydrin-positive fraction. CO2 fix- 
ation is known to occur in a number of free-living 
and parasitic protozoa, and some parasitic hel- 
minths. It appears that CO2 functions as a metab- 
olite as well as appearing as an excretory product. 

The studies on the loss of sugars from Cysticer- 
cus fasciolaris and Taenia taeniae formis in sugar- 
free solutions and the absorjjtion of glucose and 
galactose when these are available are basic obser- 
vations in work that is being continued on the 
kinetics of absorption of nutrients through the 
cestode surface. It can be expected that further 
work in this direction may lead to demonstration 
of active and selective transport. In connection 
with the cestode studies it is of interest to note that 
the data show the metabolism of larval and adult 
worms to be proportional to fractional powers of 
the body weight intermediate between those char- 
acteristic for animals with increase in metabolism 
proportial to surface area. This was an unex- 
pected finding because in tapeworms surface and 
body weight increase in the same ratio, and hence 
an increase in metabolic rate paralleling weight 
increase had been anticipated. Also, the quantita- 
tive, rather than qualitative, nature of differences 
in metabolism of adult and larval worms is note- 
worthy, since in other forms of parasites, e.g. try- 
panosomes, there are qualitive differences between 
various forms. 

Additional work on the calcareous corpuscles 
of T. taeniaeformis in comparison with artificial 
mixtures of appropriate salts indicates that the 
dolomite structure must be preformed in some way 
in the corpuscles, although they are definitely 



amorphous until heated. This is of considerable 
biological and geological interest. 

Biochemical studies on intact rat liver mito- 
chondria have shown that a reversal of DPN- 
flavin-linked oxidative phosphorylation is 
involved in the reduction of acetoacetate to succi- 
nate, with an energy transfer equivalent to one 
high-energy bond per molecule of acetoacetate re- 
duced. This energy can be supplied by one or both 
of the two terminal respiratory chain phosphoryla- 
tions without the intermediary of extramitochon- 
drial ATP. Oxaloacetic acid inhibits oxidation of 
succinate by DPN-depleted mitochondria. The 
inhibition is not of the classical competitive type 
but can be overcome by added ATP. Mitochon- 
dria inhibited by oxaloacetate gradually regain 
ability to oxidize succinate after the oxaloacetate 
is removed. These findings support concepts pre- 
viously advanced that there is a compartmenta- 
tion of substratelevel phosphorylation within 
mitochondria. This work, on basic biochemical 
pathways, is of general importance and obviously 
may be useful in explaining mechanisms for suc- 
cinate metabolism in parasites. 

Virus Potentiation by Trichinella 

The potentiation of encephalomyocarditis virus 
by Trichinella spiralis in mice is closely related to 
the size of the worm inoculum and the time in the 
worm infection that the virus challenge is given. 
A similar phenomenon, of enhanced multiplica- 
tion of Coxsackie A-9 virus in murine muscle, is 
again related to T. spiralis infection. 

The studies on Taenia taeniaeformis infection 
in mice, which seem to equate natural resistance 
with an accelerated response of acquired resist- 
ance, are of general interest. The demonstration 
of a possible immune tolerance in animals receiv- 
ing T. taeniaeformis antigen shortly after birth, 
is of considerable significance; this phenomenon 
has not been demonstrated previously in relation 
to parasites. 

Toxoplasmosis 

Toxoplasma gondii has been found in the ovaries 
and oviducts of healthy chickens. The organism 
is present in the cyst form tliat can survive diges- 
tion. The possibility that it can be found in shelled 



154 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



eggs is important in regard to the epidemiology 
of toxoplasmosis. Also, it has a technical impor- 
tance relative to the use of chick embryos for cul- 
tivation of viruses and rickettsiae. 

In sheep, resistance to congenital transmission 
of toxoplasmosis is manifest only against moderate 
challenges. No protection is manifested against 
high challenge doses. This is of interest in human 
medicine. Exposure to large numbers of Toxo- 
plasma can occur in women of particular popula- 
tion groups that customarily enjoy raw meat, and 
under such circumstances it is possible that 
habitual abortion may occur. This has been re- 
ported from Germany but not from the United 
States. 

Electron microscopy of Toxoplasma has re- 
vealed a schizogonic reproductive process, some- 
times with the production of two filial parasites 
within a parent cell and sometimes apparently 
with the production of small rosettes. A third 
process, in which two organisms are involved, may 
represent longitudinal fission or a sexual fusion. 
These new observations lend support to the con- 
cept that Toxoplasma belongs among the Sporozoa 
of the Protozoa, probably in a new sub-class along 
with organisms such as Besnoitia and Sarcocystis. 

Under the electron microscope, the formation 
and character of the cyst wall of Toxoplasma has 
been studied. The wall arises from interaction of 
the parasite and the host cell. The nature and 
development of the cyst are important in under- 
standing chronic infection. Although some activ- 
ity of cysts is evident in chronic infection, long- 
term treatment with pyrimethamine begun as early 
as three weeks after infection can produce some 
diminution in residual brain infection but cannot 
eliminate it. 

Progress has been made in the fractionation of 
ToxoplasTna antigens. Separation of hemaggluti- 
nating antigen on a hydroxyl apatite column yields 
an initial fraction that induces hem agglutinating 
(HA) antibodies but not dye test antibodies when 
injected into rabbits. This fraction has spectro- 
photometric activity indicating a high proportion 
of nucleoprotein, presumably from the cytoplasm 
of the parasite. Attempts to get a "clean" cell 
wall preparation to induce dye test antibody for- 
mation in the absence of HA antibody have thus 
far been only partially successful, in that such 
fractions have stimulated dye test antibodies more 
rapidly. If the complete separation can be 



attained, one can conclude that dye test antibodies 
are produced against the cell wall and that hemag- 
glutinating and complement fixing antibodies are 
stimulated by the intracellular components of the 
parasite. 



LABORATORY OF TROPICAL VIROLOGY 

The third year of the existence of the Laboratory 
has seen continuation of field-laboratory investiga- 
tion on viral disease at the IVIiddle America Re- 
search Unit (MARU) in the Panama Canal Zone 
and supportive laboratory studies in Bethesda, 



% 



A 



Venezuelan Equine Encephalitis (VEE) 

The first isolation of VEE virus in Panama was 
accomplished in April from specimens obtained 
from a dying Panamanian boy. Three senior 
laboratory staff members became infected as a 
result of the exposure (with virus isolation and 
serological confirmation in each case) . The entire 
MARU contingent was then immunized with live 
attenuated VEE virus vaccine. Mild reactions 
were often encountered, and the attenuated virus 
was isolated from some of the vaccinees. Several 
aspects of practical application of this still experi- 
mental preparation were thus explored. 

Epidemiologic and ecologic studies in collabora- 
tion with Gorgas Memorial Laboratory (MGL) 
of the rural community where the case had oc- 
curred, failed to demonstrate the VEE virus in 
wild, domestic or sentinel animals, or in the mos- 
quitoes collected. Serological evidence of VEE 
infection (CF, HI, NT) was established for a 
significant percentage of residents of the area. 

A remarkable urban epidemic of VEE occurred 
in another part of Panama near the site of the 
Gorgas Memorial Laboratoiy field station. Ap- 
proximately 350 cases were registei-ed during a 
two months' period. Important new information 
on the epidemiology and the clinical spectrum of 
epidemic infection with this virus is being devel- 
oped by GML and MARU. 

Vesicular Stomatitis Virus (VSV) 

Last year we reported repeated isolation of 
Indiana type VSV from phlebotomus sandflies 
collected in the course of a collaborative project 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



155 



with Gorgas Memorial Laboratory on the ecology 
of arthropod-borne viruses. Approximately 700 
hmnan sera were collected from the population of 
a town near the field station. One-fourth of the 
490 sera tested had neutralizing antibodies to 
Indiana type VSV. One-fourth of the neutraliz- 
ing sera also possessed CF antibodies. 

Interest in this virus prompted investigation of 
an outbreak of vesicular stomatitis in cattle. The 
causative agent was found to be the New Jersey, 
rather than Indiana, type of VS virus. Antibody 
patterns of humans in close or remote association 
with the infected cattle suggested that direct con- 
tact, rather than arthropod vectors, may be in- 
volved in the transmission of this infection to man. 
It would seem tliat the ecology and epidemiology 
of the two types of VSV are quite dissimilar. 

Virus From Rain Forest Arthropods 

During the second year of the study on the 
ecology of arthropod-borne viruses in the tropical 
rain forest of Panama, conducted by GML in 
collaboration with MAEU, the number of virus 
isolates was doubled : present two-year total 38-28 
from mosquitoes and 10 fromi phlebotomus sand- 
flies. The isolation rate from sandflies continues 
to exceed by far the overall isolation rate from 
mosquitoes. 

The first isolate of Indiana VSV from a non- 
sandfly source was recorded (mosquito Gultex 
nigri-palfus) . Many of our mosquito isolates 
have been identified with collaboration of the 
Trinidad and Belem laboratories of the Rocke- 
feller Foundation: six strains closely related to 
Una virus of Group A (work with four other 
similar agents has not been completed) ; one strain 
of Ilheus virus of Group B ; several members of 
the Bunyamwera group-one Guaroa and four 
Wyeomyia viruses ; Guama group, represented by 
six isolates, all from the same mosquito {Culex 
vomerifer). A few other agents remain un- 
identified. 

Attempts To Recover Viruses From Parasitic 
Mites 

Nearly 500 pools of acarines (including 25 
species of chiggers, 35 of parasitoid mites and a 
few ticks from over 350 wild vertebrates) were 
inoculated into suckling mice and hamster kidney 



cell cultures. Not a single viral agent was iso- 
lated, attesting to the unconamon presence of 
at least the recognizable viruses among the com- 
mon tropical mites. Many ectoparasite pools 
were shipped to the Eocky Mountain Laboratory 
for attempted recovery of rickettsia. One posi- 
tive isolation of the Gosoiella I>urnetii-like agent 
was made from a species of intradermal chigger, 
parasitic on the tropical spiny rat. 

Mycoses 

Repeated isolation of HisfoplasTna capsulatum 
from bat liver and spleen suspensions was a most 
important finding, providing further evidence for 
the suspected role of bats in the ecology of histo- 
plasmosis. The first human case of actinomycosis 
(cervical-facial type) caused by ActinoTrvyces 
iovis was recorded demonstrating the existence 
of this disease on the Isthmus of Panama. 

ROCKY MOWJTAIN LABORATORY 

Investigations have continued with the same 
general objectives as previously; those of one 
large group are oriented toward the zoonoses and 
arthropod-borne diseases, and those of another 
major group are chiefly concerned with problems 
related to allergy, immunology, and resistance to 
disease. Both divisions involve basic laboratory 
research, some of which could lead ultimately to 
development of applied and control measures. 
While investigations of various vector-borne 
illnesses have never actually diminished, occasional 
changes in emphasis have occurred in the last 
few years with new leads, such as provided in 
encephalitis ecology by the mosquito-garter-snake 
relationship, discovery of new foci of Powassan 
and California viruses, and variations in spotted- 
fever-like isolates. On the other hand, attacks on 
phases of such nonvector-borne disease problems 
as tuberculosis, Salmonella, pertussis, poliomyeli- 
tis, influenza, and certain mycoses have been 
intensified in varying degrees. 

Tularemia 

Wliile it was reported under Pasteurella tul<i- 
rensis last year that "the protection afforded by 



643351—62 



-12 



156 



ANNUAL REVIEW OF INTRAMXJRAL RESEARCH 



live organisms was not effective for long periods 
of time,'* mice later vaccinated with a living 
Kussian attenuated strain had a high level of 
immmiity for more than 30 weeks. 

Further comparison of isolates from different 
sources and geographical localities has 
strengthened the evidence that a "fully virulent" 
organism in North America causes severe disease 
in sheep, hares, rabbits, horses, and man. A less 
virulent form is common to North America and 
also to Europe and Asia. 

Disease of Wildlife 

Cory neb actenum fyogenes was cultured from 3 
dead mountain sheep during an epizootic and from 
1 deer and 1 elk. These findings suggest that this 
majr be a pathogen of consequence to certain wild- 
life, as well as to domestic animals. 

Three more isolations of rabies from bats this 
year bring the local total to 24. Contrary to most 
concepts, rabies can be chronic in mice. A conse- 
quent discovery Avas that recovered mice with limb 
movement impaired by rabies became heavily in- 
fested with ectoparasites. Mechanical interfer- 
ence with combing and grooming, caused by 
amputating limlbs of normal mice, resulted in 
similar excessive parasite populations. Another 
unidentified, nonfatal virus was incidentally iso- 
lated from bats and passed in tissue cultures and 
mice. 

In the absence of a usable skin test for detecting 
the protozoon, Toxoplasma microti in Microtus, 
an effective method that does not cause permanent 
injury to the animal was devised for observing 
brain lesions through a slit m the scalps of mice. 
Tolerance for this organism may result from in 
utero, or very early postnatal, infection in native 
mice. 

A fungus, Emmonsia crescens, foimd in certain 
indigenous small animals of several continents, 
may also infect man, as indicated by positive 
passive cutaneous anaphylaxis tests applied to hu- 
man serums. 



Arthropod-Borne Viruses 

Western equine encephalitis (WEE) virus was 
isolated from Culex tarsalis at Vale, Oregon dur- 
ing the midsummers of 1960 and 1961, but not in 



the months from October to May or from bird 
tissues collected during this period. The over- 
wintering virus mechanism still remains illusive. 
Recovery of WEE virus in the spring of 1961 
from captive gai'ter snakes that had been bitten 
before hibernation by infected mosquitoes is sug- 
gestive of one method of overwintering in spite 
of failure, as yet, to find naturally infected snakes. 
This lead has been a popular one and has caused 
examination of reptile-virus relationships in many 
coimtries. 

Three isolates of California virus have been 
made in the Bitterroot Valley from ticks off a 
snowshoe hare, a golden-mantled ground squirrel, 
and a chipmunk. Antibodies occurred in a low 
percentage of these local animals. This naturally 
leads to an inquiry into the role of ticks as vectors. 

An additional isolation of Powassan virus from 
ticks in western South Dakota has resulted in 
intensified ecological studies in this area with 
consequent finding of antibodies in serums from 
local chipmunks and wild mice. A human serum 
from western INIinnesota and one from central 
North Dakota (of 1,058 miscellaneous samples 
tested) showed high neutralizing indexes against 
this virus. Regional field work will be focused 
on ecologic and public health aspects of this virus, 
in view of the known significance of the related 
Eussian spring-summer encephalitis. 

Q Fever 

Field investigations this year were oriented 
toward a more objective evaluation of the poten- 
tial public health problem associated with the vast 
reservoir of infection among dairy cattle. As 
determined through continuous surveillance, even 
minor illness among persons exposed to infected 
dairy cattle could not be attributed to Q fever. 
However, 87% of persons exposed to infected 
herds have acquired antibodies detectable by the 
mouse-neutralization test and the radioisotope 
precipitation test. 

Investigations on developing a safe method of 
vaccinating humans with conventional Q fever 
vaccine were continued at Montana State Prison. 
Most of the skin-test negative volunteers (97%) 
who last year received from 1 to 3 doses of 10 
complement-fixing (CF) units of antigen sub- 
cutaneously reacted positively on a skin test given 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



157 



10 months after vaccination. However, the ag- 
glutinin response among groups of vaccinees 
varied from 45 to 91% and was directly propor- 
tional to the number of doses of vaccine received. 
Studies on a comparable group of volunteers who 
received from 1 to 3 doses of 1 CF unit of antigen 
intradermally are not complete, but results to date 
are similar to those obtained when vaccine was 
given subcutaneously. Although the resistance 
induced in human volunteers could not be chal- 
lenged, vaccination appears to have been effective, 
as judged by available immimologic and serologic 
data. 

Significant progress has been made in investiga- 
tions directed toward fractionating C. burneiii 
into its component antigens. The separation of 
nontoxic immunogenic fractions from those re- 
sponsible for inducing or eliciting the hypersensi- 
tive state remain a major objective. All antigens 
of any consequence were determined to be located 
in the cell wall. Chemical extraction of whole 
phase I rickettsias with dimethyl acetamide, di- 
methyl sulfoxide, or a combination of trichloro- 
acetic acid and phenol have yielded protective 
antigens whose toxicity and ability to induce an 
allergic response were decreased about 100-fold. 
Similar extracts of phase II organisms were non- 
immunogenic and, in preliminary comparisons of 
whole phase I and II rickettsias, phase I organisms 
were more potent vaccines. 

Rocky Mountain Spotted Fever 

Because of the results of recent studies on 
rickettsial variants and observations made possible 
by fluorescent microscopy, interest in the biology 
of spotted fever rickettsias has been rekindled. 
Particular attention has been given to the elucida- 
tion of factors which affect the natural infection 
rate among ticks and to the characterization of 
spotted fever antigen in naturally infected ticks. 
Of ticks collected in nature, up to 25% contain 
specific antigen detectable by fluorescent micros- 
copy but only 1 to 3% contain pathogenic 
rickettsias. 

The ecology of B. rickettsii appeared to be the 
same in 2 local areas which had similar natural 
features. Heaviest infestations of immature 
Dermacenfor andersoni occurred on golden-man- 
tled ground squirrels and chipmunks, and virulent 



organisms were isolated from these rodents for the 
first time west of the Mississippi. 

Heretofore, 4 variant types of R. rickettsii in 
D. andersoni have been recognized. The spectrum 
of pathogenicity of these types varies from an 
ability to cause overt disease and death to a limited 
ability to cause only immunizing, inapparent in- 
fections. From ticks collected in eastern Montana, 
many isolates of a fifth type have been recovered. 
These strains are nonpathogenic for guinea pigs 
and mice and appear to be antigenically different 
from other types of R. rickettsii. The relationship 
of the fifth type to the ecology of spotted fever 
rickettsia remains to be clarified. 

By the use of the fluorescent antibody technique, 
the gross discrepancy between the incidence of 
pathogenic rickettsias among D. andersoni and the 
prevalence of specific antigen in this tick has been 
tentatively explained by the demonstration of a 
rickettsia like organism that is noninfectious for 
chick embryos as well as for guinea pigs. This 
agent behaves like E. rickettsii because it is trans- 
ovarially transmitted by D. andersoni, has a sim- 
ilar distribution in tick tissues, and is indistin- 
guishable from R. rickettsii by fluorescent 
microscopy. Studies will be continued to de- 
termine whether this organism may influence the 
prevalence of pathogenic rickettsias among ticks 
in nature. 

Mechanisms of Allergic Phenomena 

A particular skin protein which had been altered 
by combination with a simple chemical plays an 
important role in contact allergy. "Wlien a con- 
jugate of a purified heterologous protein and 
hapten was used, antibody but not contact hyper- 
sensitivity to the hapten developed. However, 
when a conjugate of a simple chemical and a 
soluble fraction from guinea pig skin was used as 
a. sensitizing agent, contact hypersensitivity to 
the protein developed. This clearly shows that 
contact hypersensitivity, such as poison ivy rash, 
is dependent upon combination of chemicals with 
substances in the skin. 

Since the delayed hypersensitive state can not 
be quantitated by conventional tests in the skin 
of sensitized animals, an attempt was made to use 
the cornea as a site for measuring response to the 
antigen because this tissue is not vascularized. In 



158 



AJSnsrUAL REVIEW OF INTRAMURAL RESEARCH 



animals sensitized with a conjugate of protein and 
hapten, the reactions in the cornea occurred only 
to the protein moiety, although circulating anti- 
body to the hapten and associated allergic re- 
actions could be demonstrated in other tissues. 
In the corneal response, which is primarily cellu- 
lar polymorphonuclear leucocytes from surround- 
ing tissue and blood vessels were chemotactically 
attracted to the antigen. 

Although delayed shock is thought to be a sys- 
temic manifestation of delayed hypersensitivity, 
the reaction apparently is dependent also upon 
circulating antibody. In guinea pigs sensitized 
with hapten-protein conjugates, only injection of 
the protein causes the systemic delayed reaction 
when only delayed hypersensitivity to the conju- 
gate is present. However, after antibodies have 
appeared, intraperitoneal injection of the conju- 
gate causes a brief initial hyperthermia followed 
by a distinct hypothermia and lymphopenia typi- 
cal of delayed shock. At this time delayed shock 
can not be precipitated by the injection of protein 
alone but it can be produced by injection of hapten 
combined with a heterologous protein. 

Although many immunologic reactions seen in 
comparative studies of allergic phenomena in new- 
born and adult guinea pigs were similar, the de- 
layed response evoked by intradermal injection 
of antigen could not be elicited before 14 days 
after birth. This unresponsiveness is attributed 
to the inability to excite a normal inflammatory 
response, but the basis for this inertia has not been 
fully clarified. 

To date, these aiad related studies on tolerance 
and the effect of hormones and vitamins on de- 
layed allergy support the thesis that delayed hy- 
persensitivity and antibody formation are but 
phases of the same process. Fundamental knowl- 
edge of the mechanisms involved should provide 
some insight into the study of allergic phenomena 
associated with infectious and autoimmune 
diseases. 

Endotoxins 

Although the exact chemical composition of en- 
dotoxin has not been defined, the biological activ- 
ity of endotoxin complexes, contrary to previous 
conceptions, cannot be associated with the lipid 



fractions, and the active principle is probably a { 
polysaccharide. 

In further studies, in which endotoxin com- 
plexes were depolymerized by treatment with di- 
lute acid, biological activity, including pyrogenic- 
ity, tumor damage, lethality, and immunogenicity, 
was associated with the size of the molecule. 
When depolymerization had progressed to the 
extent that the haptenic imits were about Viooth 
the size of the original endotoxins, all biological 
activity was eliminated. These studies indicate 
that one of the major requirements for endotoxin 
to elicit host reactions is a macromolecular com- 
plex of critical size. 

Other methods of depolymerizing and recom- 
bining the haptenic units will be used to clarify the I 
relationship between molecular size and biologic 
activity. 

Morphological Elements of Microorganisms 

Through collaboration with designers and en- 
gineers of a firm specializing in the manufacture 
of laboratory instruments, a self-contained refrig- 
erated cell-fractionator patterned after the pro- 
totype made at KML was tested and developed for 
the market. The machine is designed so that bio- 
logical materials can be subjected to any selected 
jjressure up to 60,000 psi and then by sudden re- 
lease of material through .a cooled orifice, organ- 
isms or tissue cells can be disintegrated without 
denaturation of any of the cellular components. 
During the past year, 12 guest workers and visitors 
came to EML specifically for the purpose of using 
this instrument or learning more about it. Since 
cellular material, varying from the size of small 
bacteria to large mammalian cells, can be com- 
pletely disintegrated without denaturing any of 
the chemical constituents, this instrmnent will have 
varied and extensive application in driverse prob- 
lems in bio-medical research. 



Radioisotope Precipitation 

The development and application of a radio- 
isotope precipitation teclmique (RIP test) to serol- 
ogy of virus diseases and the study of virus syn- 
thesis received major consideration. This tech- 
nique is based on the principle that radioactive 



I 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



159 



particulate antigen (25 m^ to 300 m/t in diameter) 
sensitized by specific antibody is agglutinated after 
species-homologous antiglobulin is added to the 
reaction. No removal of excess globulin is neces- 
saiy as it is in conventional Coombs type tests 
where visible precipitate, rather than removal of 
radioactivity from suspension, is the indicator. 
The test has been standardized so that it possesses 
a high degree of sensitivity and specificity. In the 
determination of specific antibody, the test is 30- 
fold more sensitive than the tissue-culture ne^^trali- 
zation test for poliovirus and 100-fold more sen- 
sitive than agglutination or complement-fixation 
tests for Q fever. By the use of an inliibition-type 
modification, it is possible to detect as little as 0.1 
to 0.001 ug of antigen. The inhibition-type modi- 
fication has been found particularly useful in 
studies of viral metabolism wherein it is necessary 
to detect specific viruses, viral components, and 
enzymes which may be stimulated by ENA and 
DNA of plant and animal viruses. 



LABORATORY OF GERMFREE ANIMAL 
RESEARCH 

Interest in the use of germfree animals in an 
increasing variety of research projects continues 
to grow. More and more, members of our staff 
are asked to lecture on germfree animal research, 
teclmiques, and applications, at scientific sessions 
and before university staff. At present, in addi- 
tion to collaborative work with the staff of other 
laboratories in our own Institute, we are engaged 
in projects with scientists of the Cancer Institute, 
CDC, and three universities. It is anticipated that 
this type of cooperation will continue, especially 
when the cooperating groups have materials, tech- 
niques, and tests which will be helpful to us, but 
which would not be worthwhile for us to attempt 
to attempt to set up. 

The possibilities offered by germfree, virus-free, 
or virus-defined animals for serologic, tissue cul- 
or virus-defined animals for serologic, tissue cul- 
ture and perhaps pharmacologic standards will, it 
is felt, receive increasingly more attention, and 
has been the subject of discussion among scientists 
working with germfree animals. 



Susceptibility by Sex 

In the collaborative studies with the Laboratory 
of Parasitic Diseases the early impression that 
the sex effect known to occur in certain helminth 
infections in conventional animals does not occur 
in germfree animals has been corroborated several 
times. Whatever the mechanism involved, the 
lack of a flora reverses the effect normally shown 
in conventional females. The latter are poor hosts 
for Nematospiroides dubius, for example. We are 
currently studying mono-infections with individ- 
ual species of intestinal bacteria in an effort to see 
if we can pinpoint this interesting relationship. 
Studies by others working with mono-infected 
animals have shown that individual species of 
bacteria can bring about a specific physiologic ef- 
fect. Among these relationships has been a dem- 
onstration of the effect of bacteria on steroid com- 
pounds in vivo. 

Dietary Effect on Hehninth Infections 

An effect of diet on the course, and duration of 
certain helminth infections has been observed. 
This effect appears to be holding up in germfree 
animals as well as in those reared under conven- 
tional circumstances. These infections do not do 
as well in an animal on a semi-synthetic diet of 
casein, carbohydrate, vitamins, etc., as they do on 
the stock Purina-type laboratory animal feed. 
However, the germfree state appears to compomid 
the difference. 

"Natural" Antibodies 

In studies on the occurrence of so-called "nat- 
ural" antibodies in uninoculated animals, both 
germfree and conventional, several interesting 
findings have been made. We have found that 
mice from a colony which has been reared for at 
least 7 years free from viable E. coU, /S. typhosa, 
etc., show about the same reactivity to Gram-nega- 
tive organisms and their products as do conven- 
tional animals. No differences were obtained in 
levels of bactericidal antibody, resistance to endo- 
toxin and phagocytic response. This is of particu- 
lar interest inasmuch as it had been postulated by 



160 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



others (in theoretical discussions of modes of ac- 
tion of endotoxin) that germf ree animals might be 
considerably more resistant of endotoxin because 
of their lack of exposure to Gram-negative organ- 
isms. It would appear that even though there are 
no viable, metabolizing, Gram-negative organisms 
in the animal's intestinal tract, repeated ingestion 
of small doses of the heat-stabile endotoxin ma- 
terial inevitably present in the diet is sufficient to 
stimulate the animals to form "natural" antibodies. 
A similar situation seems to prevail with respect 
to antibodies or antibody-like activities against 
Staphylococcus antigen. In a variety of tests in- 
volving serum-gel-diffusion, agglutination, and 
fluorescent antibody techniques, uninoculated ani- 
mals not harboring any bacteria that could be de- 
tected still demonstrated staphylococcal antibodies. 
It was noted that the germfree animals did not 
develop these "natural" antibodies at as early an 
age as did the conventional animals. Animals 2-3 
months of age were negative, whereas those 7-8 
months of age showed considerable activity. 
Again, one must conclude that prolonged ingestion 
of small amounts of heat-stabile staphylococcal 
antigenic material (it has recently been demon- 
strated that such substances exist) can stimulate 
the formation of antibodies. These findings point 
to the source of some of the "natural" antibodies 
that occur in uninoculated animals. Eventually, 
we may have to resort to animals raised on soluble 
diets of small-molecule materials prepared under 
conditions which (hopefully) would preclude the 
presence of these, apparently, ubiquitous heat- 
stabile antigenic materials. 

Mouse Colony Free of Virus 

In our continuing search for evidence of the 
presence of viruses in our mouse colony, the results 
continue to be essentially negative for most of the 
viruses tested. In samplings of well over 125 ani- 
mals there has been the suspicion of the presence of 
only one virus in a few instances — Reo 3. At- 
tempts to isolate the agent from bedding, etc., 
from colony units have not been successful. 
Among other viruses tested for which negative re- 
sults were obtained are included mouse adenovirus, 
poloyma, GD VII, hepatitis and K virus. These 
studies will continue until we are reasonably sure 
of the viral state of our colony. However, thus 



far, it would appear that our germfree mice are 
free of some of the more common mouse viruses 
plaguing viral studies in conventional animals. 

Thyroiditis in Guinea Pig 

In collaborative studies with the Laboratory of 
Immunology, thyroiditis has been produced in 
some guinea pigs as early as 5 days after immuni- 
zation. This constitutes the earliest recorded ob- 
servations of this autoimmune disease in experi- 
mental animals. The lesions are found in all 
animals 16 days after immunization, and at 7 weeks 
the disease is severe and extensive in all animals. 
This has persisted as long as 6 months, at tlie end 
of which time some decrease in severity has been 
found. In studies on mechanisms involved in this 
autoimmune disease, antibody levels, as well as 
tests for delayed hypersensitivity to thyroid ex- 
tract, have been followed. Findings thus far sug- 
gest that the presence of the disease is correlated 
with delayed hypersensitivity, but not necessarily 
with circulating antibodies. Inasmuch as auto- 
allergy is felt, at present, to be the underlying 
process in several human diseases, information de- 
rived from these studies as experimental models 
can be of potential importance. 

Trauma in Amoebic Infections 

In continued studies aimed at an understand- 
ing of the role played by bacteria in aiding 
Endamoeba histolytica to establish infection and 
produce lesions in the intestine, efforts were di- 
rected at trying to determine whetlier trauma or 
damaged mucosae were essential. As we reported 
previously, it was possible to get some chronic- 
type lesions in the intestinal wall of germfree 
guinea pigs with amoebae that were prepared by 
techniques which apparently resulted in more vig- 
orous organisms than had been used in the past. 
However, most of the lesions seemed to occur at the 
site of the inoculation. Recently we have tried 
inoculations via the terminal ileum whereby the 
amoebae were delivered into the cecum at some dis- 
tance away from the puncture wound. Under 
these conditions the number of germfree animals 
developing lesions in the cecum was significantly 
reduced. Thus, at least in the case of the type of 
lesion obtained in the absence of a bacteria flora, 






NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



161 



trauma or some frank tissue break appear to be 
necessary for the amoebae to establish infection. 

Second Generation Guinea Pigs 

Some further studies on the biology of germf ree 
guinea pigs were conducted. The lack of bacteria 
seems to have more profound and diverse 
deleterious effects on guinea pigs than on other 
laboratory animal species that have been reared. 
We were able to obtain two litters of second gen- 
eration germfree animals. Only rarely and re- 
cently has this been possible. The growth rate 
and general physical condition of these young was 
better than has been experienced with Caesarean- 
derived animals. The fact that they received 
maternal milk perhaps may explain their better 
performance. 

Germfree Chickens 

In further studies to ascertain activities of the 
intestinal flora, it was found that 6-week-old germ- 
free chickens did not contain valeric acid in their 
intestinal tracts. This substance is a common con- 
stituent of gut contents in conventional chickens 
and, therefore, these findings indicate that it arises 
from microbial fermentation. Of further interest 
was the fact that chromatographic analyses of 
gastro-intestinal material from germfree chickens 
failed to reveal any fermentation acids in the crop. 
However, lactic acid was demonstrated to be pres- 
ent throughout the lower GI tract in amounts 
which are normally found in conventional birds. 
This rather unexpected finding in the absence of a 
demonstrable flora must be explored further. 



LABORATORY OF INFECTIOUS DISEASES 

The work of the virus and epidemiology sections 
continued to be aimed at better definition and un- 
derstanding of numerous human and animal virus 
infections, particularly of their roles as etiologic 
agents in respiratory disease, cancer and birth 
defects. In other words, the chief concern is to 
find the true natural history of viruses, and define 
their importance in various disease states. In or- 
der to do this it has been necessary to develop ap- 
propriate tests and survey methods and in 1961 



we were able to achieve comprehensive surveys of 
viral infections not only in man but also special 
studies of domestic and commensal animals. We 
have continued field and clincal studies in collab- 
oration with research persoimel of the Bureau 
of Medicine and Surgery, U.S. Navy ; D.C. Chil- 
dren's Hospital Eesearch Foundation; the D.C. 
Welfare Department; the National Institute of 
Neurological Diseases and Blindness ; the National 
Cancer Institute; and the Laboratory of Clinical 
Investigation of the National Institute of Allergy 
and Infectious Diseases. 



Respiratory Virus Disease 

Previous annual reports and publications have 
documented the key role of LID scientists in the 
discovery of adenoviruses, parainfluenza viruses 
and respiratory syncytial viruses, and in defining 
their importance in causing respiratory illnesses, 
especially in children. In 1961 the role of these 
agents in causing 35 to 45 percent of the acute 
respiratory illness of infants and children was 
confirmed. In addition, the contribution of the 
enteroviruses (Coxsackie and ECHO viruses) to 
the acute respiratory syndrome was partly eluci- 
dated. Studies of outbreaks of Coe virus (Cox- 
sackie A-21) in military recruits, Pett (Coxsackie 
A-24), Coxsackie B 3 and 5 viruses suggest that 
these enteroviruses (and new unclassified ones 
similar to the British Salisbury viruses) will soon 
be found to contribute significantly to the overall 
respiratory disease problem. 

Eaton Agent is PPLO 

The most significant finding since the beginning 
of our studies of Eaton's "virus" (Primary Atyp- 
ical Pneumonia) was the demonstration in 1961 
that it is not a virus but a PPLO (pleuropneu- 
monia-like organism) which can be grown in 
synthetic media. 

This discovery is exciting for three reasons. 
First it opens the door for the development of 
much simpler techniques for diagnosing primary 
atypical pneumonia. Secondly, it provides a ready 
source of material for preparing a preventive 
vaccine, and thirdly, it suggests that additional 
serologically different PPLOs can be expected to 
turn up as causes of acute respiratory illnesses. 



162 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



The fact that pneumonia due to the Eaton agent 
responds readily to tetracycline therapy and the 
fact thta PPLOs are known to be generally very 
susceptible to broad spectrum antibiotics fur- 
nished previously undreamed of possibilities in 
the therapy of respiratory illnesses. 

Enteroviruses in Acute Respiratory Disease 

The enteroviruses (Poliovirus, Coxsackie and 
ECHO viruses) are well known as causes of cer- 
tain specific illnesses such as poliomyelitis, her- 
pangina, pleurodynia, aseptic meningitis and myo- 
carditis. Less well appreciated are their impor- 
tance in causing acute upper respiratory illnesses. 
It is evident from our long term studies of Junior 
Village children (where we observed nearly 2,000 
persons infected with many different enterovi- 
ruses) that acute respiratory illnesses often with 
fever represent the most common clinical mani- 
festations of most enteroviruses. Eecently using 
more sensitive tissue culture isolation procedures, 
we have encountered numerous additional viruses 
having the properties of Coxsackie A viruses. The 
most common group of similar isolates are related 
to Coxsackie A-24 Pett virus which is related 
to Coxsackie A-24 virus. 

Information on the role of enteroviruses as 
etiologic agent of adult respiratory illness, partic- 
ularly the common cold syndrome has been derived 
chiefly from outbreaks of ECHO 28 (2060 virus) 
reported at different times chiefly from Chicago 
by Magabgab several years ago and in 1961 by 
Hamre and the outbreaks of Coxsackie A-21 in 
Marines observed last year. It appears that vi- 
ruses can be recovered from about 20 to 25 percent 
of adults with mild respiratory illnesses, thus 
providing new dimensions for study of the "com- 
mon cold." 

Antibiotics in Acute Respiratory Disease 

"We completed studies on the value of tetra- 
cyclines in the treatment of primary atypical pneu- 
monia due to Eaton's (PPLO) agent in adults 
and on the value of oral penicillin in preventing 
acute febrile respiratory illnesses in children. The 
first study of pneiunonia which was carried out in 
Marine recruits at Parris Island (in collaboration 
with the Bureau of Medicine and Surgery, U.S.N.) 



showed that declomycin, a synthetic of tetra- 
cycline, was very effective in the treatment of 
primaiy atypical pneumonia, reducing signifi- 
cantly the duration of fever, pneumonitis and 
hospitalization. 

The daily use of oral penicillin for nearly a year 
in a proportion of children at Junior Village 
failed to significantly alter the overall illness rate 
despite the fact that hemolytic streptococcal in- i 
fections were virtually eliminated from the throats ! 
of those in the test group. Only during brief 
outbreaks of several typable hemolytic strepto- 
cocci was it possible to show any measurable 
beneficial effects from the drug. No effect was }| 
observed on viral infections. 



Respiratory Viruses in Human Volunteers 

Many studies of numerous respiratory agents 
(viruses and PPLO) in volunteers are scheduled 
to be carried out in cooperation with the Labora- 
tory of Clinical Investigation and the Federal 
Bureau of Prisons. To date studies of the patho- 
genesis of several parainfluenzas, RS, and Coe 
(Coxsackie A-21) viruses, and Eaton's PAP 
agent grown in tissue cultures have been com- 
pleted. All were found to cause acute respiratory 
illnesses. Eaton's agent produced not only pri- 
mary atypical pneumonia but also middle ear 
infections ; in the latter case providing one of the 
first specific clues to the cause of nonbacterial 
serious myringitis. 

One of the more interesting findings from the 
volunteer studies was the fact that many of the 
virus infections produced illnesses despite the 
presence of pre-existing antibodies ; but the anti- 
bodies also were responsible for the general mild- 
ness of the illnesses observed. 

Studies in volunteers of Coxsackie A-21 virus 
(a Coe-like strain isolated from a military re- 
cruit) revealed that growth of this enterovirus 
was almost entirely restricted to upper respira- 
tory tract. This is interesting since other Cox- 
sackie A-21 strains are commonly found in the 
stools of children. Carefully designed volunteer 
studies provide a wealth of high order informa- 
tion on host-parasite relationships virtually un- 
attainable in any other way. Hence they will be 
continued and expanded. 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



163 



Vaccine Development 

One of the chief purposes of the volunteer study 
program is to test live viruses for attenuation and 
for inclusion as candidates in experimental vac- 
cines. Of course, killed vaccines can also be tested 
in volunteers — the eiScacy of the vaccine can be 
measured by challenge with a pathogenic variant 
of the virus as previously done by LID scientists 
in developing the first effective adenovirus vaccine. 



Sero-Epidemiology of Human Viral Diseases 

During 1961 antigens against more than 100 
common viruses (6 myxoviruses, 28 adenoviruses, 
ES, 3 reoviruses, 59 enteroviruses, and 10 mouse 
viruses) were evaluated in the micro-CF and 
micro-HI test systems. This program represents 
collaboration between the cerebral palsy study 
group of NINDB and all of the research units in 
the Virus and Epidemiology Sections of LID. 

Extensive serologic-epidemiologic surveys are 
now in progress on the prevalence of antibody 
responses against more than 100 viruses in women 
giving birth to abnormal babies, and in children 
and adults with acute respiratory illnesses, neu- 
rological diseases, and cancer. The prevalence of 
specific adenovirus and myxovirus antibodies in 
sick children at Junior Village and Children's 
Hospital is now under study, thus adding a new 
dimension to our knowledge of the natural history 
and pathogenesis of these agents. 

Similarly serological surveys of these popula- 
tions have revealed new and wholly unexpected in- 
formation on the enteroviruses. Previous data 
from Junior Village which suggested that enter- 
oviruses were important causes of acute respira- 
tory illness, and that this may be the most frequent 
illness caused by enteroviruses, was confirmed by 
serologic studies of patients from D.C. Children's 
Hospital. These tests showed in 1961 that entero- 
virus infections in children with acute respira- 
tory disease were eight times as frequent as in 
age-matched control children without respiratory 
illness. 

Cancer Viruses 

Studies of animal cancer viruses have proceeded 
satisfactorily both in the field and in the labora- 



tory. Perhaps our most important function is 
to focus attention on the importance and feasibility 
of studying the occurrence and behavior of cancer 
viruses in nature, to help develop laboratory tools 
which make such studies possible, and to continue 
to help define the extraneous viruses in cancer virus 
study systems which are producing almost insuper- 
able obstacles to the development of reliable in- 
formation about cancer viruses. 



Natural History of Polyoma Virus 

The natural cycle of polyoma virus was found 
to be well established in Mus musculus living in 
farm industry ecologies in Maryland and in In- 
diana. There is extensive evidence that the age- 
old association of the house mouse first mentioned 
in relation to grain storage in the Mediterranean 
area (recorded in ancient Egyptian and Greek 
records and the Old Testament) now persists in 
modern grain storage facilities, on farms and in 
central depots all over the world. Although Miis 
rruusculus is now indigenous to all parts of the 
world, it originated in southern Asia and the Mid- 
dle East, from whence it was carried along lines 
of trade to other parts of the world reaching north- 
ern Europe and England by 900 A.D. and becom- 
ing well established in the New World only within 
the last two centuries. Studies in southern Eng- 
land and nationwide surveys in the United States 
show that infestation of grain storages are ex- 
tensive, almost universal, and frequently very in- 
tensive. For instance, a recent 29 state U.S. survey 
revealed an average count of 80 mouse pellets per 
peck (about 15 lbs) of shelled com. 

LID studies in the field to date show that poly- 
oma virus infection could be demonstrated in Mus 
musculus on 4 of 15 Maryland dairy farms, in 
6 of 6 Maryland cereal grain mills and 3 of 4 
Indiana grain mills. Polyoma virus was isolated 
from cereal grains used chiefly for livestock feed 
(but also for laboratory mouse and human con- 
sumption) wherever such grains showed objective 
evidence of mouse contamination and where the 
mice were found to be positive for polyoma. TMs 
was not surprising, since infected mice are known 
to excrete large amounts of virus for long periods 
in the urine and infestation with polyoma-positive 
mice was very heavy in most of the ecology studies. 
Thus, as reported last year, the polyoma cancer 



164 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



virus is widely distributed in all the ecologies in 
which connnensal house mice live — in laboratories, 
in production colonies of laboratory mice, in 
densely populated urban tenements, an in rural 
environments. 

Extraneous Viruses in Cancer-Virus Study 
Systems 

This problem has if anything become worse. 
Additional viruses were found in production 
colonies of laboratory mice, including PVM, new 
strains of hepatitis and Eiley's plasma agent. For 
instance, Eiley's agent, together with reovirus 3 
and hepatitis virus turned up successively in our 
passage lines of Moloney's leukemia. 

On the credit side we have been able to develop 
satisfactorily in vitro survey tests for mouse hep- 
atitis, PVM, Theiler's virus, and LCM, thus 
adding these to our test battery which already in- 
cluded tests for polyoma, K virus, mouse ade- 
novirus and mouse salivary gland virus. Thus by 
working with commercial producers of laboratory 
mice, we hope to achieve rather soon supplies of 
mice that are free of infections with these agents. 

Immunological studies of mouse reoviruses 
showed them to be indistinguishable from those 
isolated previously from man, cattle and monkeys. 
An interesting sidelight was the demonstration 
that Stanley's encephalohepatitis virus is identical 
with reovirus type 3 and that the various other 
hepato-encephalitis agents isolated from mice and 
described by Cheever and others are serologically 
related to MHV and other strains of hepatitis 
virus. 



SPF and Germfree Mice 

The obvious answer to the background noise 
jiroblem is the use of susceptible SPF (specific 
pathogen free) mice and germfree mice. The 
main difficulty with the latter is that they simply 
are not available in large numbers. The SPF mice 
as suggested above, are of value only when they 
can be kept in isolated virus-proof quarters, which 
currently are not available in our virus building. 

We have tested two methods of isolation — both 
with considerable success. The first method made 
use of isolated house trailers which are chemically 
sterilized between experiments. These have been 



very useful for studies of polyoma virus in such 
field specimens as mouse tissues, excreta and mouse 
contaminated grain. 

Recently, successful experiments have been per- 
formed m the Lobund plastic isolators. Although 
these require much attention and high caliber 
animal attendants, they may well be the answer 
to the production of pedigreed, clean and certified 
cancer virus pools — a prime requii'ement in cancer 
virology just as it is for respiratory virology. 



Fungus Disease 

Mj'cology studies have followed a multidiscipli- 
nary and multifacted research program. In the , 
field the work is focused on isolation and identifi- I 
cation of new fungi and on ecologic studies on 
various fungi known to be pathogenic for man. 
The studies of new fungi are done in collaboration i 
with the hospitals and diagnostic laboratories ; i 
the ecologic studies in collaboration with the 
Rabies Control Unit, Trinidad, West Indies. New 
antimycotic drugs and antibiotics are tested for 
efficiency and safety, in collaboration with the 
Medical Physiological Bacteriology Section of 
LID and with the Laboratory of Clinical Inves- 
tigation. The clinical and field studies are sup- 
ported and supplemented by basic woi-k in labora- 
tory on the physiology, toxicity and immunology 
of pathogenic fungi. The search continues for 
serological methods capable of recognizing my- 
cotic infections in humans and in various animal 
hosts, and for unpi'oved tecluviques designed for 
growing and identifying fungi. FTistoplasma 
capsulatwn was isolated in Washington, D.C. for 
the first time from a congested urban area, from 
soil adjacent to houses in Trinidad, W.I., which 
harbor bats, and from soil under roosting sites of 
starlings. 



Penicillinase 

A higlily interesting observation concerning 
staphylococcal penicillinase has been made. It 
seems probable that this enzyme is a non-specific 
cyclic peptidase, splitting various cyclic peptides 
to straight chain compomids — this observation 
offers at least a partial answer to the hitherto 
disturbing question of the role and function of 
penicillinase in bacteria in their natural habitat. 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



165 



Under nornaal ecological conditions, it is highly 
unlikely that bacteria ever comes into contact 
with penicillin, hence the value of the possession 
of penicillinase to the total economy of the cell 
has been dubious. By offering the possibility that 
penicillinase is primarily a specialized peptidase, 
its role is conceivably enlarged to a more impor- 
tant function in cellular metabolism. 

Antibacterial Marine Waters 

Significant observations have been made on the 
problem dealing with the antibacterial activity of 
marine waters. It is now quite clear that the ac- 
tivity is due to a large organic molecule with mo- 
lecular weight greater than 10,000 rather than due 
to the inorganic ion content of the sea water. This 
observation, obtained as a result of exhaustive 
dialysis experiments, settles in large part a contro- 
versy of 75 years amongst biological oceanogra- 
phers. Partial isolation and purification of the 
active factor has been obtained. Further, the 
activity seems to be quite specific for gram- positive 
bacteria, thus offering an attractive hypothesis in 
explanation of the fact that the vast majority of 
hacterial genera m the oceans are gram negative. 
The activity is always present in ocean water, 
varying only quantitatively and this fuiding an- 
swers in part the hitherto disturbing observation 
that activity of raw sea water varies temporally 
and seasonally. From the point of view of isola- 
tion and purification of an antistaphylococcal fac- 
tor, particularly active against penicillinase-pro- 
ducing staphylococci, this is, of course, decidedly 
advantageous. 

Staphylococcus Iron Metabolism 

Highly significant observations have been dis- 
covered this year dealing with metabolic differ- 
ences occurring between iron-deficient and 
iron-sufficient strains of Staphylococcus aureus. 
The degree of iron depletion or enrichment was 
controlled by the ratio of free Fe- to iron-bound 
siderophilin in the medium of growth of the 
organisms. Using radioactive tracers, it is now 
quite clear that iron-deficient cells lack the ability 
to metabolize the six carbon of glucose when com- 
pared with iron-sufficient cells and further that the 
deficient cells also have decreased ability to in- 



corporate carbon into protoplasm. These obser- 
vations are a highly important beginning in a 
project desigTied to understand the biology of 
staphylococci. Since production of the various 
staphylotoxins presumably mirrors the overall 
metabolic processes of the cells and since, as indi- 
cated above, iron plays such a highly important 
role in metabolism, the hope remains that by 
juggling iron availability in vivo (and this can be 
done by proper use of siderophilin), one will be 
able to render invading staphylococci less toxic. 
In the development of the above thesis, it has 
been essential to devise methods for purification 
of physiologically active siderophilin and for 
assaying such activity. This has finally been done 
by various ingenious biochemical techniques and 
the problems can now be attacked forthrightly. 



Cell Division in Streptococci 

Study of the M antigen of Group A, beta- 
hemolytic streptococci had yielded a surprising but 
highly important dividend of basic significance. 
A controversy which has been going on for 
decades over whether synthesis of the cell wall 
in dividing bacteria occurs by random intercala- 
tion or at specifically polarized growing loci has 
been all but settled, at least for the Group A 
streptococci. Using, alternately, periods of 
growth in culture media with and without fluores- 
cein-labelled homologous anti-M protein, the issue 
has been definitely decided in favor of the latter 
hypothesis, i.e., polarization. The observation is 
of significance to all studying cytokinesis and 
morphogenesis in bacteria and fungi as well as 
to those interested in cell-wall metabolism in gen- 
eral. Interest, of course, in cell walls has been 
high in recent years since many important inhib- 
itors, including penicillin, presumably act by 
virtue of inhibiting cell-wall synthesis. 

Further improvement of the "long-chain" test 
for streptococcal type specific antibody has been 
made and its simplicity and accuracy appear likely 
to make it a most valuable tool for the answering 
of many clinical and epidemiological studies. 
Currently, it is being used in the study of pre- 
and post-isolation sera from a type 12 streptococ- 
cal outbreak among institutionalized nursery- 
school-age children. 



166 



ANNUAL REVIEW OF INTRAMTJRAL RESEARCH 



The mechanism of the long-chain reaction has 
been under investigation and the possibility exists 
that the phenomenon is a form of agglutination 
requiring bivalent antibody and may not be 
enzymatic as suggested by other workers. 

Antibacterial Substances in Mollusks 

Of vast potential import is the isolation, char- 
acterization and partial purification of antibac- 
terial and antiviral substances from mollusks, 
particularly oysters. Two fractions, decidedly 
different in behavior on columns, but both pre- 
sumably glycoproteins, have been found to be 
active against bacteria, including staphylococci 
and viruses (polyoma and influenza). The 
significance of this is obvious for an effective 
chemotherapy of small virus infections is of great 
moment. 

Hydrogenomonas 

Marked advances have been made in an under- 
standing of the electron transport of Hydro- 
genomonas and as a consequence, relying on the 
thesis of comparative biochemistry, the main 
energy-yielding systems of manmialian tissues. 
The system in the autotrophic Hydrogenomonas 
is ideal for these studies since it is, in distinction 
to systems in all other forms studied, at least 
partially soluble. This fact obviates the extreme 
difficulty of working with particulate and hence 
highly complex material. A further indication 



of the relative simplicity of the complex in this 
organism has been the finding that it contains only 
one cytochrome. It now seems obvious that flavo- 
proteins are active in this system in the transport 
of elutions and judicious thesis can be presented 
for the formation of reduced pyridine nucleotides 
via the hydrogenase system. However, a new 
intermediate, different from others heretofore de- 
scribed, apparently is involved in the formation 
of reduced pyridine nucleotide. This, of course, 
has vast theoretical ramifications and would indi- 
cate important differences between bacterial and 
human energy-yielding systems and might well 
indeed be a basis for selective attacks on bacteria 
by antibacterials. 

Eosinophilic Meningitis 

Field investigations in French Polynesia un- 
covered an extensive outbreak of eosinophilic 
meningitis. The epidemiologic evidence suggests 
the disease is associated with the consmnption of 
raw fish infected with a helminth. Wliile these 
cases were being investigated, two patients died 
of a similar condition in Hawaii. From the brain 
of one of these, young adult nematodes identified 
as Angiostrongylus cantonensis, the lung worm of 
the rat were found in the brain substance and in 
the meninges. Intermediate hosts of this parasite 
are terrestrial slugs and snails. In spite of the 
clinical similarity of cases in French Polynesia 
and Hawaii, there are epidemiologic differences 
which remain to be explained. 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC 

DISEASES 



BASIC RESEARCH 

Introduction 

The review by Laboratories and Clinical 
Branches of the scientific accomplishments of the 
National Institute of Arthritis and Metabolic 
Diseases for the calendar year 1961 is herewith 
presented. To the casual reader, this summary 
may appear to be a discoordinated series of indi- 
vidual reports of the activities of many individual 
scientists. A more intimate contact with the par- 
ticipating scientists reveals currents and cross- 
currents running through the several administra- 
tive segments of NIAMD and, indeed, through 
the several Institutes of N.I.H. Big areas of 
common interest are apparent. The origins of 
such interests, the nature of the infective foci and 
the mechanism of the spread is a rewarding epi- 
demiologic study. 

Genetics 

Perhaps the most prominent such epidemic is 
the disseminated interest evident this year in 
phenomena related to genetics. As one reads the 
attached summary it is clear that almost every 
segment of NIAMD has been contaminated with 
an interest in one or another aspect of the genetic 
problem. 

In the patient, a considerable list of genetically 
transmitted defects continues to be studied. These 
include phenylketonuria, alkaptonuria, cystinosis, 
gout, galactosemia, cystic fibrosis and the glyco- 
genoses. In the intact animal, genetic studies of 
the transmission of joint disease and of obesity 
are described. The chromosome, which is the ulti- 
mate visible unit of genetic transmission, is the 
subject of special scrutiny. Abnormalities in the 
number and morphology of chromosomes are being 
correlated with abnormal phenotypes. Genetically 
transmitted defects in the amino acid sequences 
of individual proteins which are under study in- 



clude variations in hemoglobins and in lacto- 
globulins. 

The information transfer which is fundamental 
to the genetic process rests upon the structure and 
biosynthesis of the nucleic acids. Accelerating 
activities in the area of the chemical structure and 
physico-chemical properties of ENA will be f oimd 
described herein. The properties of these com- 
pounds in solution as well as in the solid state are 
being examined by the most sophisticated methods. 
The enzymes which generate these polymers and 
degrade them are being purified and studied. 

Of unusual interest are the mechanisms whereby 
information contained in nucleic acid structure is 
transferred into the amino acid sequence of pro- 
tein. A remarkable step forward in the under- 
standing of this so-called "code" has been taken 
in the year just completed. Working with cell- 
free protein synthesizing systems, NIAMD scien- 
tists have deciphered the first words of tliis crypto- 
gram. Thus "polyuridylic acid" in the language 
of ENA means "-phenylalanyl-" in the language 
of polypeptides. AVe like to consider this accom- 
plishment, like that of ChampoUion in unscram- 
bling the cartouches of the Eosetta Stone, as 
initiating a new era. 

Enzyme-Catalyzed Reactions 

Another area of scientific activities which tran- 
scends administrative divisions is the study of 
regulatory mechanisms. It is becoming increas- 
ingly apparent that one of the advantages which 
results from the near-universal enzyme catalysis 
of biochemical events is the opportunity thus pro- 
vided for regulation. In various portions of the 
succeeding report will be found references to such 
mechanisms. Thus, analysis reveals that the effect 
of hormones of the anterior pituitary gland upon 
the end-organs, the effects of posterior pituitary 
extracts upon mammary gland, are mediated 
through effects upon enzymes. The specific effects 

167 



168 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



of certain steroids upon aldehyde deyhdrogenase 
have been carefully studied and documented. 
Feedback and repression mechanisms have been 
studied in the regulation of histidine synthesis 
and evidence has been presented that the enzymes 
involved in urea synthesis are adaptive. The 
enzymes concerned with insulin destruction in 
the liver are likewise regulated by feedback 
mechanisms. 

Perhaps the most completely analyzed example 
of regulation of a specific enzyme is that of hepatic 
glutamic dehydrogenase. It has been clearlj^ 
shown that this enzyme exists in at least two ag- 
gregation states. The larger aggregates are active 
against one amino acid, the smaller units are active 
against another amino acid. The process of ag- 
gregation-disaggregation is reversible and is 
exquisitely sensitive toward various reagents such 
as estrogens, pyridine nucleotides, certain amino 
acids, and various other compounds of biological 
interest. 

Intrinsic to the nature of enzyme catalysis is 
the capacity for the great variability which is an 
essential condition for survival. Examples are 
now coming to light in increasing numbers 
wherein we can define, with ever greater precision, 
how this variability is accomplished. 

General 

Representatives of our Institute have spent pro- 
longed periods, in the past year, in laboratoiies in 
England, Scotland, Switzerland, France and Ja- 
pan. Visiting scientists from 14 countries are 
presently in residence at Bethesda. 

Educational activities are growing. The Ee- 
search Associate program continues to provide 
training in laboratory science to selected physi- 
cians and this operation is clearly attracting the 
most capable people of appropriate age group and 
background. In the opinion of those participat- 
ing, this program has become an important part of 
the way of life in NIAMD. 



LABORATORY OF EXPERIMENTAL 
PATHOLOGY 

Cell Structures in Aging 

Studies of certain cytoplasmic bodies demon- 
strable by histochemical techniques in aged 



animals have been extended. As a result the wide- 
spread distribution of cytosiderin and of oxida- 
tion-aldehyde-fuchsin reactive particles has been 
determined in retired breeder animals of four ro- 
dent species. The epithelial inclusions containing 
Prussian blue reactive iron, according to the most 
likely interpretation, derive from breakdown 
products of the electron transport system. This 
cytosiderin and the morphologically similar bodies 
in the same or different histologic sites which can 
be visualized by an oxidation-aldehyde-fuchsin 
stain, may be examples of de Duve's lysosomes, 
since in the instances thus far investigated they 
also contain hydrolytic enzymes. Cells containing 
cytosiderin inclusions examined in the electron 
microscope by Dr. Bruce Wetzel have shown vari- 
ably vacuolated dense bodies with dark granules 
possibly similar to ferritin morphologically. 

Cytogenetic Studies 

During the past year studies have been contin- 
ued on chromosomes of neoplastic cells for a criti- 
cal evaluation of the de BOVEEI concept of 
carcinogenesis. Additional studies have been con- 
cerned with a comparison of the relationship be- 
tween chromosome constitution on the one hand 
and human hereditary diseases and de^"elopmental 
defects on the other. A project investigating the 
biosynthesis of polio virus by euploid fibroblast 
cells of non-neoplastic origin has been completed 
as has also a collaborative study of certain abnor- 
mal human sera with mammalian chromosomes 
employing the Coon's fluorescent antibody 
technique. 

Degenerative Joint Disease 

Studies of the pathogenesis of degenerative 
joint disease and of the descriptive pathology of 
human rheumatism have been extended. Osteo- 
arthritis was found to occur less frequently in 
female than in male STR/IN mice. Orchiectomy 
did not reduce the amomit of osteoarthritis ; thus, 
the arthritis enhancing effect of maleness was pre- 
sumably due not so much to a testicular contribu- 
tion as to an absence of an ovarian one. 

Studies of the occurrence and nature of sclerosis 
of blood vessels in normal human joints are near- 
ing completion in collaboration with Dr. Stanley 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



169 



Elmore of the N.H.I. These lesions have not been 
described previously ahhough they are very com- 
mon. The significance of the findings, for the 
most part, lies in their recognition as non-rheu- 
matic changes. In addition, however, they are as- 
sociated witli small areas of infarction in the 
pulvinar acetabuli of older individuals. It is 
suggested that this may be a cause of a presently 
imrecognized syndrome of hip pain in elderly in- 
dividuals who apparently have no arthritis. 

An interesting by-product of studies of joint 
disease in mice has been the recognition of geneti- 
cally governed obstructive uropathy in male STE/ 
IN mice. The lesions were prevented by castration 
and never occurred in females. They are a major 
cause of mortality in this group of animals and 
jDrobably unrecognized in certain others as well. 

Hematology 

It has been shown that erythropoietine promotes 
the differentiation of stem cells into erythroid ele- 
ments. The hypothesis was advanced based on 
studies on irradiated animals that the turnover 
rate of the stem cell compartment is governed by a 
negative feedback; differentiation of stem cells, 
then, serves as a stimulus for stem cell production. 
Intense erythroid stimulation whether due to 
severe anemia, hypoxia, or erythropoietine leads to 
the production of macrocytes, which have a 
shortened life span. The cause of the macrocytosis 
is as yet unproven but is most likely explained by a 
shortening of the emergence time together with 
skipping of divisions. 

The growth potentiality, function and life span 
of the small peripheral blood lymphocyte have 
been the subject of controversy for many years. 
Using H^ thymidine it was demonstrated that in 
culture the small lymphocyte of the peripheral 
blood cell undergoes transformation after which 
it begins to synthesize DNA and divide (MacKin- 
ney, Stohlman and Brecher). Using H^ thymi- 
dine, it was conclusively demonstrated that the 
small lymphocyte has a life span of more than 
100 days while the large lymphocytes have a life 
span of 60 days. (Brecher) 

Hemoglobin 

The genetic abnormality in Hb I, which pro- 
duces a hemolytic anemia, is in the a chain, where 



there is a substitution of aspartyl for lysyl in the 
16th amino acid residue. A unique optical prop- 
erty of sickle cell hemolysates was demonstrated in 
these hemolysates. There is a reversible increase 
in the positive Cotton effect of 3.5 fold suggesting 
that there is a dynamic conformational change of 
the hemoglobin molecule. (Dr. Murayama) 

The constant for the mercapto-mercapto inter- 
action in the a chain of fetal hemoglobin is similar 
to that of the a chain of adult hemoglobin but the 
constants of the y chain differ from those of the 
P chain of adult hemoglobin (fetal hemoglobin 
a2y2, adult hemoglobin a2;82) . The energy barrier 
due to steric hindrance in fetal hemoglobin is 2 
kilocalories/mole. This value is somewliat less 
than that of adult hemoglobin, indicating a 
"loosening" of the molecule, which results in a 
greater affinity for Oo as well as for Ag-I- ions. 
(Dr. Murayama) 

Although the formation of Heinz bodies and the 
inhibition of heme enzymes by phenylhydrazine 
have been observed for many years, the mechanism 
of action of this compound has not been clear. It 
now apears that the active molecule is not phenyl- 
hydrazine itself, but is an unstable oxidation 
product, monophenyl, diimide. (Dr. Itano) 

Histochemistry 

The first conclusive evidence that carotid body- 
like tissue in the human contains norepinephrine 
and the first report of hypertension-producing 
carotid body-like tumor lias been reported from 
this laboratory. Proof of the presence of 
norepinephrine in this chemoreceptor tumor was 
demonstrated by histochemical methods and veri- 
fied by chromatographic and biochemical analysis. 
Norepinephrine was localized to unique and char- 
acteristic argentaffin cells bounding the non-argen- 
taffin cell nests of normal carotid bodies and carot- 
id body tumors. Evidence from this study 
suggests that the argentaffin cells in chemoreceptor 
tissue originate from the neural crest and partici- 
pate in the sympatho-adrenal system, joerhaps as 
local modulators of baroreceptor response or as 
norepinephrine stores responsive to local or gen- 
eralized tissue anoxia. These findings also sug- 
gest that the hypertension of hypoxia may be at 
least partially of local reflex origin and imply a 
direct regulative effect on vasomotor control by 
chemoreceptor tissue. (Dr. Glenner) 



I 



170 



ANlSrUAL REVIEW OF ENTRAMUBAL RESEARCH 



A study of enzyme systems involved in the syn- 
thesis and breakdown of tissue proteins has re- 
vealed by histochemical methods the presence in 
the islets of Langerhans of the guinea pig of an 
enzyme hydrolyzing a-glutamyl peptides. An 
enzyme of this type has not previously been de- 
scribed either biochemically or histochemically. 
Also described for the first time histochemically is 
the precise tissue localization of a y-glutamyl 
transpeptidase enzymic activity believed to be of 
significance in the synthesis of specific proteins, 
and a peptidase activity in peripheral and cen- 
tral nervous system myelin, active in the mainte- 
nance of protein structure in nervous tissue. By 
these histochemical methods defects of protein 
metabolism in specific cells can be detected and 
their relationship to pathologic processes deter- 
mined. (Drs. Glenner, Hopsu, McMillan and 
Spatz) 

Efforts have been directed toward development 
of methods for identification of specific muco- 
substances. The histochemical classification of 
mammalian mucins thus developed includes types 
not previously distinguishable histologically as 
well as certain mucopolj^saccharides not recog- 
nized biochemically. Histochemical methods 
have been fairly well established for differentiat- 
ing sulfated from non-sulfated acid mucins. A 
technique for identifying most of the latter as 
sialomucins has been devised in collaboration with 
Dr. L. Warren. In combination with other pro- 
cedures this technique distinguishes sialomucins 
which are digestible by Vibrio cholerae sialidase 
from non-digestible sialomucins. A third type of 
acid mucopolysaccharide has been recognized 
which lacks sialic acid and sulfate esters and con- 
tains an unknown acid group. Histochemical 
methods have also been worked out for demon- 
strating periodate unreactive sulfomucins (e.g. 
heparin and chrondroitin sulfates in mast cells and 
cartilage and sulfomucins in some glands and gob- 
let cells) as well as procedures for localizing bio- 
chemically unrecognized, periodate reactive 
(hexose containing) sulfomucins in various glands 
and goblet cells. Application of these methods 
to characterization of mucins in human tumors 
has shown that benign and malignant lesions of 
the breast and tumors in patients with pseudo- 
myxoma peritonei contain sialomucins, that mixed 



tumors of the major salivaiy glands secrete peri- 
odate unreactive sulfomucins and mucoepider- 
moid tumors of the oral minor salivary glands 
secrete a periodate reactive sulfomucin. (Dr. 
Spicer) 

A histochemical method has been worked out 
which employs an acid dye in differentiating basic 
proteins according to their relative basicity. It I 
is thought that in this method the dye specifically ' 
combines with e amines or guanidino groups of 
proteins and that the staining of these two groups 
can be differentiated. In its application the pro- \ 
cedure has visualized histone type protein with 
unique histochemical properties in chromatin and I 
nucleoli. It also reveals the presence of a similar ' 
protein ( ?mucohistone) in sites containing acid 
mucopolysaccharides such as goblet cells and | 
mucous glands. (Dr. Spicer) 

Human Pathology 

The opportunities offered to this laboratory 
through consultative and diagnostic studies of 
surgical and autopsy specimens from the Division 
of Indian Health and other facilities of the Pub- 
lic Health Service have continued to stimulate 
interest in aspects of geographic and environ- 
mental pathology. Following visits to a ninnber 
of the Indian Hospitals, the quality of the ma- 
terial submitted for study has improved. There 
has also been a substantial increase particularly 
in the nmnber of autopsy specimens submitted. 
Problems related to sarcoidosis, to diabetes, to 
dietary hemosiderosis, to cholecystitis, and to 
atherosclerosis and its complications are of par- 
ticular interest. Consultative services to two 
Korean Charity Hospitals has resulted in the 
collection of 37 cases of fatal Pneumocystis carinii 
infection in children. 

In addition to the research projects summarized 
separately, certain scientists in other laboratories 
received advice from members of our staff, par- 
ticularly in pathologic anatomy. Our histo- 
pathological preparation unit also took part in 
this cooperative effort by cutting and staining 
tissue sections from about 2,000 animals this year 
for seventeen investigators not in laboratories of 
NIAMD. 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



171 



Hypersensitivity 

Rat marrow cultures are used to study in vitro 
the mechanism of the interaction between rat 
marrow cells and mouse lymphocytes sensitized to 
rat marrow. The interaction is in the realm of 
delayed hypersensitivity and may aid in elucidat- 
ing the mechanism involved in graft rejection. 
(Drs. Demopoulos and Gesner) 

Another study dealing with the generalized 
Schwartzman reaction indiiced in rabbits by two 
injections of bacterial endotoxin is being con- 
ducted. Prominent features of this reaction in- 
clude altered vasomotor reactivity, leukocyte 
damage, and intravascular coagulation. Since 
the basophilic leukocyte is believed to contain 
histamine, 5-hydroxytryptamine and heparin, 
substances which may effect vascular tone and 
blood coagulation, the possible role played by cir- 
culating basophilic leukocytes in the Schwartzman 
reaction is being examined. (Dr. Horn) 

Immunocheinistry 

In collaboration with Dr. Marian Webster a 
study has been completed of the antigenicity of 
human kallikreins from urine and pancreas after 
inoculation into rabbits. Precipitin reactions 
were readily demonstrated with homologous anti- 
sera by the Ouchterlony technique and in test 
tubes. Eabbit antiserum to hmnan kallikrein in- 
hibits the vasodilator activity of human kallikrein 
injected into dogs, but fails to precipitate or in- 
hibit either dog or hog pancreatic kallikrein. 

A study of the localization of glyceraldehyde-3- 
phosphate dehydrogenase with fluorescent anti- 
body to the enzyme has been completed. The 
enzyme has been demonstrated in high concentra- 
tion in mitochondria surrounding the I band of 
the wing and leg muscle of the cockroach. Further 
studies with tissue cultures of human and chicken 
myoblasts have demonstrated the presence of this 
enzyme in specific mitochondria close to the nu- 
cleus while other mitochondria are completely 
negative. Studies on sections of mouse and rat 
kidney have shown the specificity of the enzyme 
for certain cells and specific areas of the tubules. 

A study of the antigenicity of prolactin in 
rabbits included precipitins in gels and in test 
tubes. These studies have been coordinated with 



Drs. Condliffe and Bates with fractionations of 
prolactin. 

Marrow Cultures 

A new technique for culturing human and rat 
marrow cells is employed for the purpose of study- 
ing in vitro transmissibility of leukemias. The 
cells are grown at the bottom of a closed miniature 
well with a restricted oxygen supply. This slows 
the mitotic rate enough to permit the cells to dif- 
ferentiate. Survival of cultures is thus greatly 
prolonged offering a tool for the study of leukemo- 
ffenesis in vitro. 



Melanomas 

It was previously found that phenyllactic acid 
inhibited the growth of pigmented melanomas but 
not that of non-pigmented melanomas. Studies 
are continuing to determine further the effect of 
phenyllactic acid and of other tyrosinase inhibitors 
on the growth of pigmented and non-pigmented 
melanomas in mice. 



Nutritional Deficiencies 

Pathologic studies on conventional and germ- 
free animals given various deficient diets are con- 
tinued. A recently completed study demonstrated 
that supplementation with selenimn compomids 
markedly delays the onset and decreases the in- 
cidence of hepatic necrosis in conventional rats 
fed a diet based on 4% casein as the protein source. 
Studies are currently in progress to evaluate the 
ability of selenium compounds to prevent this 
lesion in germ- free animals. 



Serum Enzyme Changes After Stress 

Previous studies showed that large doses of 
catechol amines or a 4-hour exposure of dogs to 
hypoxia (32,000 feet altitude) caused, in addition 
to hyperglycemia, a transient sharp rise in serum 
glutamic oxalacetic transaminase (SGOT), serum 
glutamic pyruvic transaminase (SGPT), sermn 
lactic dehydrogenase (SLD) , serum alkaline phos- 
phatase (SAkP), and serum aldolase (SAld). 
Studies with adrenergic and ganglionic blocking 
agents suggested that the hyperglycemia produced 



L 



172 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



by hypoxia was due to the released of catechol 
amines from the sympathetic adrenal system and 
that the rise in serum enzymes after catechol 
amines or hypoxia was due to an increase in cel- 
lular permeability. To shed further light on the 
mechanisms causing rise in serum enzymes and 
the relation between stress and the release of 
catechol amines, serum enzymes were studied in 
animals subjected to other types of stress. 

In one study, dogs were exposed to a simulated 
altitude of 32,000 feet for 4 hours 5 days weekly 
for 7 weeks. They showed a continuing gradual 
increase in SGOT and SLD values during the 7 
weeks, while SGPT stabilized by the 2nd week at 
slightly above initial control values. SAkP and 
SAld values stabilized after the third week at 
2-3 times the initial values. After cessation of ex- 
posures, values dropped sharply during the first 2 
weeks and approached normal in 6 weeks. A sub- 
sequent single 4-hour exposure to 32,000 feet 
evoked the usual transient sharp rise in serimi 
enzyme values, but not the typical hyperglycemic 
response, observed before acclimatization. These 
studies indicate that the alterations in cellular 
permeability are reversible. Eesidual pathologic 
lesions, attributable to the repeated exposures, may 
account in part for the slow restoration of normal 
enzyme values. 

After exercising rats for 16 hours a transient 2- 
fold increase in BUN and SLD, a 4-fold increase 
in SGOT and SGPT, and a 6-fold increase in 
SAld was found. SAkP declined 30%. IS^early 
all the rats showed moderate to severe fatty 
changes in the liver, kidney and thigh muscles and 
slight fatty changes in the heart. About one- 
fourth showed a few foci of inflammation and 
necrosis in the muscle. 

The effect of cold was studied in dry and wet 
rats exposed 16 hours to 1.7 and — 5° C and 5 
hours on two successive days to 1.7° C respec- 
tively. There was a transient significant rise in 
SGOT, SAld and BUN", most pronounced and 
persistent in the wet rats, which showed a more 
profound hypothermia. Concurrently, there was 
a transient decrease in mean hematocrit, a deple- 
tion of liver and muscle glycogen and adrenal 
lipid, and a marked leukocytosis with a relative 
decrease in lymphocytes. Fatty changes were 
noted in the liver and, less frequently, in the 
kidney and heart. 



It is postulated that hypoxia, prolonged exercise 
and exposure to cold increase serum enzyme levels 
by increasing cellular permeability, permitting 
cellular enzymes to escape and accumulate in the 
blood. These findings are of practical importance 
since they indicate that serum enzyme elevations 
need not be due to myocardial infarction or other 
serious organic disease, and that exposure to vari- 
ous physical stresses must be coiisidered in the 
differential diagnosis of such diseases. 

Pneumocystis Carinii Infection 

A second focus of frequent pneiunocystis carmii | 
infection in small children in Korea was estab- 
lished during this year. From this new location, 
massive infection with Pneumocystis carinii was 
foimd in 18 out of 25 fatal cases sent to the labo- 
ratory for diagnosis. 

Our attempts to propagate the organism in cor- 
tisonized germ-free rats and mice after intrapul- 
monary injection of material obtained from heav- 
ily infected conventional rats have failed. Con- 
centrates of organisms ax-e now being prepared by 
washing and careful digestion of lung tissue from 
donor rats and by exposing such cyst suspensions 
to feeder cultures. 

The only important information which our ex- 
periments in germ-free rats and mice appeai-s to 
have produced is that such rats and mice are in- 
deed free of pneumocysts in spite of prolonged 
injections of cortisone and antibiotics which would 
have produced severe pulmonary involvement by 
this organism in conventional rats within a cor- 
responding period of time. 



LABORATORY OF BIOCHEMISTRY AND 
METABOLISM 

Carbohydrate Metabolism 

Polymers 

The polymer, alginic acid, contains a repeating 
unit consisting of D-mannuronic and L-gului'onic 
acid. Continuing studies on the pattern of its 
enzymic degradation have led to the elucidation of 
a new pathway for the bacterial utilization of 
uronic acids. The initial monomeric reaction 
product, 5-keto-4-deoxy-D-mannuronic acid, was 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



173 



found to react with a DPNH-linked dehydrogen- 
ase to form 2-keto-3-deoxy-D-ghiconic acid. This, 
in turn, was phosphorylated in the presence of 
ATP and subsequently cleaved to yield pyruvic 
acid and triose phosphate. The enzymes, alginase 
and ketodeoxygluconic acid dehydrogenase, have 
been purified and their properties examined. 

Work carried out in Switzerland in the labora- 
tory of V. Prelog relates to structure determina- 
tion of Avilamycin, an antibiotic elaborated by 
Streptomyces viridans. It consists of an aglycone, 
the partial structure of which lias been deter- 
mined, and 8-10 monosaccharide units. Two of 
the sugar components were characterized as 
6-deoxyhexoses. The third sugar varies from one 
batch of Avilamycin to another. Some prepara- 
tions contain L-lyxose, a sugar not heretofore re- 
ported to occur naturally. Others contain an 
0-methylpentose. 

Small Molecules and Coenzymes 

Further studies on the biosynthesis of L-fucose 
by Aerohacter aerogenes have substantiated the 
finding that GDP-4-keto-6-deoxy-D-mannose is 
an intermediate in the formation of this sugar. 
The GDP-heptose reported last year has now been 
isolated and completely characterized as GDP-D- 
glycero-D-mannoheptose. In addition, the enzy- 
mic synthesis of this compound has been demon- 
strated to be due to a pyrophosphorylase present 
in yeast which catalyzes the reaction of GTP and 
the heptose-1-phosphate to form GDP-heptose and 
inorganic pyrophosphate. A tentative identifica- 
tion of thymidine diphosphate N"-acetyl-D-f ucosa- 
mine fi'om C. violacewn has led to an attempt to 
demonstrate enzymic synthesis of this nucleotide. 
Also, an enzyme system from S. paratyphi A has 
been found to synthesize a new sugar nucleotide, 
cytidine diphosphate glucose. This may be a pre- 
cursor of the dideoxyhexose, paratose, which is 
one of several unusual sugars present in the sur- 
face antigenic lipopolysaccharides of certain 
microorganisms. 

An investigation of the nucleotide bound sugars 
in hen oviduct was undertaken in an attempt to 
demonstrate the existence of an iduronic contain- 
ing nucleotide. During the course of this work, 
a new class of nucleotide-linked disaccharide was 
discovered and identified as UDP-N-acetyl-glu- 
cosamine-6-phosphate-l-galactose. This material 



was isolated in pure form and subjected to a 
sequential enzymic degradation accompanied by 
recovery of the reaction product at each step. 
From the same source, a family of AMP-sugars 
has been found to exist as a labile complex. This 
material is being currently studied. 

An alternate pathway of L-ascorbic acid catab- 
olism in molds has been demonstrated whereby a 
new 3-keto hexonic acid (S-keto-D-idonic acid) 
is formed and rapidly decarboxylated to yield 
L-xylulose. This mechanism differs markedly 
from that found in mammals which has been 
shown previously to result in the formation of 
two pentonic acids, L-xylonic and L-lyxonic. 

Biosynthesis 

Thiamine 

The formation of thiamine phosphate is cata- 
lyzed by an enzyme from baker's yeast discovered 
in this laboratory, thiamine phosphate pyrophos- 
phorylase. This enzyme catalyzes the condensa- 
tion of the two ring moieties according to the 
following equation : 



Mg 



.++ 



' 'pyrimidine" — PP+ " thiazole" — P; 
thiamine— P+PP 

The reaction is readily reversible, having an ap- 
parent equilibrium constant of 6 at the pH opti- 
mum, 9. 2. Procedures have been devised for the 
enzymic determination of each substrate. The 
crystalline cyclohexylamine salt of the pyrimidyl 
pyrophosphate has been prepared and the acid 
lability of the allylic pyrophosphate ester linkage 
has been studied. 

Lipid Synthesis 

The determination of the absolute rates of lipid 
synthesis in liver by means of proton incorpora- 
tion has been undertaken. It has been shown that 
when palmitic acid is synthesized from acetate 
12.2 protons should be incorporated per molecule 
synthesized. On the other hand, the synthesis of 
palmitate from acetyl-CoA derived from glucose 
requires the participation of 15.9 protons per mole- 
cule. Fatty acid synthesis from acetyl-CoA de- 
rived from fatty acid catabolism utilizes 16 pro- 
tons per molecule synthesized. Thus in the ab- 
sence of exogenous acetate 16 protons per molecule 



174 



AJSnSrUAL REVIEW OF ESTTRAMURAL RESEARCH 



of fatty acid synthesized should represent the 
number used in determining fatty acid synthesis 
in complex systems. Consistent with the fact that 
glucose and/or fatty acids contribute equally with 
acetate in the formation of acetyl-CoA, 14 protons 
per molecule fatty acid synthesized are taken up 
when liver slices are incubated with 5 X 10"^ M 
acetate. It has been calculated that 0.4 /imole of 
fatty acid is synthesized per gram liver per hour. 

Regulatory Mechanisms and Hormones 

Insulin 

Studies relating to the enzymic degradation of 
insulin by mamalian liver have been continued 
using perfused liver and broken cell preparations. 
Neither ACTH nor glucagon are effective com- 
petitors of insulin-Ii=i degradation by perfused 
rat liver. This is to be contrasted to the substan- 
tial competition which these protein hormones ex- 
hibit in liver homogenates. Thus, while the same 
enzyme system present witliin the liver cell may 
carry out the ultimate degradation of a number 
of protein hormones, access to this system in in- 
tact cells may be limited by binding to specific loci 
on the cell membrane. This conclusion is consist- 
ent with previous evidence for the binding of in- 
sulin-I^" on or in the cell membrane as an obli- 
gatory first step in the catabolism of this hormone 
by liver. 

A highly purified liver enzyme preparation, 
homogeneous according to electrophoretic and 
sedimentation studies, was found to catalyze the 
reductive cleavage of the disulfide bonds of in- 
sulin in the presence of glutathione as the hydro- 
gen donor. Studies on the physiological role of 
this enzyme could implicate it in the over-all 
"insulinase" system for the hepatic breakdown of 
insulin. Of significance also would be its possible 
relation to the mechanism of action of insulin, 
prior reduction of insulin being postulated by 
some to precede physiological action. 

Humoral Agents in Uremia 

Studies on the chemical nature of the humoral 
agents which accumulate during uremia and the 
chemical basis for the changes in cerebral function 
in uremia have been extended. The assay for an 
agent in human uremic serum which alters the 
metabolism of nervous tissue is based on the fact 



that normal rat brain cells when suspended in 
uremic serum generate greater yields of C^Oa 
from glucose C" than do the same cells suspended 
in normal serum. The agent is unaffected by tryp- 
sin, trichloracetic acid or heat, is partly dialyzable 
and is removed by cation and anion exchange 
resins. 

Pyrophosphatase 

Many critical, reversible, biosynthetic reactions 
also result in formation of inorganic pyrophos- 
phate and for this reason it becomes of interest to 
study the properties and possible control mecha- 
nisms for enzymes that split inorganic pyrophos- 
phate. There are at least two such enzymes in 
mammalian liver. One is present in the soluble 
fraction of the tissue. It requires Mg*+, has a pH 
optimum of about 7.8 and resembles yeast pyro- 
phosphates. The other occurs in microsomes, has 
a low pH optimum and does not require Mg**. 
This particulate enzyme has transphosphorylation 
as well as hydrolytic activity. Glucose, mannose, 
fructose, and galactose have been found to act as 
phosphate acceptors, while ribose, creatine, and 
maltose and inactive. 

Galactosemia 

Eight subjects who had typical symptoms of 
congenital galactosemia in infancy have been 
studied. The bed blood cells of each are essen- 
tially devoid of P-gal uridyl transferase enzyme 
and are incapable of oxidizing galactose-1-C^'' to 
C^^02. However, two of these subjects, one pre- 
pubertal and one post-pubertal, are, at present, 
near normal in their capacity to oxidize galactose 
in vivo. Thus the usual erythrocyte diagnostic 
test for this disease does not necessarily reflect the 
state of the whole organism. Furthermore, it ap- 
pears that in some galactosemic patients a tissue 
(or tissues), other than red cells, may acquire a 
pathway for galactose metabolism in the course 
of maturation. The biochemistry and genetics of 
this remain to be elucidated. 

Steroid Horm,ones 

More and more evidence is accumulating on the 
remarkable effects of steroid hormones on enzyme 
structure and enzymic catalysis. With respect to 
structure, the method of intensification of fluores- 
cence of boimd pyridine nucleotides was employed 



NATIONAIi INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



175 



to determine the state of aggregation of the glu- 
tamic dehydrogenase molecule under various con- 
ditions. Evidence was found for the fact that 
hormones do unfold the molecules, exposing addi- 
tional pyridine nucleotide bind sites, and from this 
evidence, the approximate molecular weight of 
the DPNH binding units was determined. The 
method of light scattering was also used and con- 
firmed the fact that there is a rapid equilibrium 
between the sub-miits and the aggregate, and that 
the position of the equilibrium is influenced by the 
steroid hormones. 

The bacterial induced enzyme /3-galactosidase 
was used to investigate the effects of steroids on 
the synthesis of this enzyme, and it was found that 
there was a stimulatory effect by the hormones on 
this synthesis. This effect was most readily ob- 
served when normal enzyme synthesis was in- 
hibited by puromycin. 

A possible regulatory f miction for steroids was 
discovered in their curious effects on aldehyde 
dehydrogenase. A partially purified aldehyde 
dehydrogenase for rabbit liver has been found to 
be inliibited by one group of steroids and either 
stimulated or inhibited by another group, depend- 
ing on the concentration of aldehyde. Since 
further efforts to purify the enzyme were unsuc- 
cessful, the less direct approach was utilized of 
examining the effect of reagents which are known 
to alter the configuration of proteins. A low con- 
centration of urea, for example, was found to pro- 
duce an effect similar to diethylstilbestrol 
(stimulation) with high aldehyde concentration — 
inhibition with low aldehyde concentration, and 
at a higher concentration of urea, one similar to 
progesterone (inhibition). It thus appears that 
steroids may act on the enzyme system by altering 
the secondary or tertiary structure of the enzyme 
molecules, although lack of effect of steroids on the 
sedimentation velocity suggest that this influence 
is not accomplished through dissociation of the 
enzyme into subunits. 

Another enzyme system subject to control by 
steroids involves the oxidation of retinene. Eeti- 
nene is oxidized irreversibly to vitamin A acid by 
a pyridine nucleotide-linked aldehyde dehydrogen- 
ase present in liver, kidney, and small intes- 
tine. Diethylstilbestrol, dehydroisoandrosterone, 
estrone, and cortisone stimulate the reaction rate 
at optimal substrate levels ; diethylstilbestrol also 



inhibits when substrate is below 3 X 10"* M. Pro- 
gesterone, desoxycorticosterone, testosterone, and 
androsterone inhibit at all substrate levels. The 
Km for retinene is 1.4X10"^ M. The energy of 
activation for retinene is 14.6 kilocalories per 
mole, and is descreased to 11.9 by stilbestrol and 
increased to 20 by progesterone. The stimulation 
produced by stilbestrol is reversed by dialyzing 
away the hormone. 

Nucleic Acids 

Striking progress was made in the purification 
of polynucleotide phosphorylase from Micrococcus 
lysodeikticus and now, for the first time, the en- 
zyme is available in high purity, and with a rigid 
primer requirement. There is a nearly absolute 
requirement for oligonucleotides of the type 
pApApA both for making polymer and for ca- 
talyzing the exchange reaction between inorganic 
phosphate and nucleoside diphosphate. This work 
will make possible a really definitive study of 
polynucleotide phosphorylase and also has prac- 
tical value in the synthesis of polyribonucleotide 
chains all of which are built on small primer units 
as a nucleus. Such materials has already been used 
in studies on the genetic code (see below) . 

Alkaline phosphomonoesterase from E. coli and 
a number of other non-specific phosphatases were 
purified, and each of them contained a low but 
definite ribonuclease activity. By many criteria 
this nuclease activity was shown to be part of the 
same protein enzyme. This led to the generaliza- 
tion that non-specific phosphatases have an intrin- 
sic component of ribonuclease activity, that is 
differentiated from true nucleases by being inhibi- 
ted by low concentrations of inorganic phosphate. 
Such 'a significant generalization is satisfying, but 
has unfortunate consequences for the problem of 
nucleotide sequence in E.NA. All schemes for nu- 
cleotide sequence depend on sequential removal of 
terminal phosphate from ENA, and non-specific 
phosphatases have been widely used for this pur- 
pose, due to the mistaken impression that they 
were safe reagents. 

Polyguanylic acid of large size than formerly 
available has the interesting property of being 
just about completely resistant to every enzyme 
tried on it. Presumably this reflects a high degree 
of interaction of guanylic acid units along the 



176 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



polynucleotide chain, thus protecting the polymer 
from enzymatic attack. Incorporation of the base 
analogues, azaguanine, into KNA of Bacillus 
cereus has been studied and doubt is cast on 
earlier reports concerning the way in which this 
base is arranged in the nucleic acid molecule. 
Polyazaguanylic acid has been made with poly- 
nucleotide phosphorylase and its properties 
studied. 

We realize today how important is the secondary 
structure of nucleic acids. Specific hydrogen 
bonding interrelationships provide the mechanism 
for hereditary transmission, and this accounts for 
the interest in physical studies, such as the follow- 
ing : The determination of tautomeric forms (i.e., 
the positions of the hydrogen atoms responsible 
for hydrogen bonding) in polynucleotide helices 
has permitted this structural feature to be used in 
order to rule out a recently proposed, crystal- 
lographically feasible alternative to the Watson- 
Crick model for DNA. Where products of the 
interaction between different nucleic acids differ 
only in the positions of the exchangeable hydro- 
gens on the bases, knowledge of these positions of 
attaclnnent can be used as a criterion of macro- 
molecular structure. 



Cell-Free Protein Synthesis 

A cell- free system for protein synthesis was ob- 
tained from E. coli and stabilized so that the en- 
zyme extracts could be stored frozen without 
undue loss of activity. This system has been used 
to provide a cell-free assay for messenger ENA and 
to show that polyuridylic acid can serve as a spe- 
cific carrier of information for the incorporation 
of phenylalanine and polycytidylic acid for pro- 
line. Various copolymers have since been found 
active for the incorporation of other amino acids 
into polypeptide structure. It is clear that the 
genetic code is yielding to an experimental ap- 
proach. It was also found that ribosomes from 
one species can make protein from another species 
if the proper conformational ENA is added, and 
that "phenylalanine soluble-ENA" is the inter- 
mediate through which phenylalanine is incor- 
porated into protein. 



Lysogeny 

Biochemical studies of the metabolic events oc- 
curring during bacteriophage infection have been 
fruitful in many laboratories. Because of its 
great genetic interest, infection of E. coll K,2 
cells by lethal mutants of the phage A was inves- 
tigated. The DNA polymerase present after in- 
fection shows minor differences when compared 
to the DNA polymerase of uninfected cells. How- 
ever, the deoxyribonuclease activity of E. coli Kj. 
is increased about 10-fold by bacteriophage in- 
fection. This increase in nuclease activity is under 
examination as a possible control mechanism in 
the induction of lysogenic organisms. Addition- 
ally, it has been found that the glucose repression 
of A production in K,; is due to the faulty adsorp- 
tion of the bacterioplaage by cells which are grown 
in glucose. Presumably a receptor site for A has 
been affected. 

Histidine Biosynthesis 

Continuing investigation of the biochemistry of 
histidine formation and the control mechanisms 
regulating this pathway (feedback inhibition) 
has resulted in the isolation of the enzyme catalyz- 
ing the first step in histidine biosynthesis. ATP 
and 5-phosphoribosylpyrophosphate are condensed 
by this enzyme to form N-l-(5-phosphoribosyl)- 
ATP. The enzyme has been purified approxi- 
mately 1,700 times over wild-type levels and ex- 
hibits a Ki for L-histidine of 5 X 10"= M at pH 
7.5. The inhibition is strongly pH dependent with 
a ]dK at 9.0 suggesting that inhibition of the en- 
zyme by histidine is dependent upon the presence 
of a charged a-amino nitrogen. I 



Model Compounds 

The enzyme, thiooxidase, has been isolated from 
the fungus, Pirlcularia onjzae^ and purified to the 
stage where it sediments as a homogeneous protein 
in the ultracentrifuge. The preparations catalyze 
two reactions: 1. The oxidation of compounds 
bearing — SH on ethylenic or aromatic carbons 
to their corresponding disulfides. 2. The oxidation 
of catechols. Based on constant ratios of the two 
activities throughout fractionation, elution pat- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



177 



terns from resins and sedimentation patterns upon 
nltracentrifugation, it is concluded that thiooxi- 
dase and catalase are functions of one enzyme. 



LABORATORY OF CHEMISTRY 

Carbohydrates 

The chemistry of 2-deoxy-D-ribose has been ex- 
plored in a variety of directions thanks to the 
ready availability of this sugar through a simple 
preparation developed earlier by H. W. Diehl. 
A key intermediate used earlier by E. K. Ness for 
the synthesis of deoxynucleosides has now been 
obtained in crystalline form by E. K. ISTess. The 
substance, 2-deoxy-3, 5-di-(9-^-nitrobenzoyl-D- 
ribosyl chloride is a representative of a compara- 
tively little known class of sugar derivatives and, 
because of its utility in nucleoside synthesis, has 
been examined in some detail. Eeactions in- 
volving replacement of the chlorine atom are, as 
expected, non-stereospecific. Means have been 
found, however, to influence the proportions of 
stereoisomeric products in a useful manner and 
this finding was of importance in the synthesis of 
the two anomei-ic 2-deoxy-D-ribofuranose phos- 
phates. The chloride was found to decompose 
readily to furfuryl ^-nitrobenzoate, a reaction 
reminiscent of the formation of kinetin from 
deoxyribonucleic acid. 

The 3-deoxyaldohexoses having the ardbino and 
ribo configurations are of current interest in cer- 
tain biochemical studies but the recorded synthetic 
methods for making these compounds are cum- 
bersome and difficult. A method was devised by 
H. B. Wood, Jr., for making both of them in a 
simple two-step synthesis from 2-deoxy-D-ribose. 

A very labile sugar phosphate of intermediary 
metabolism is 2-deoxy-D-ribose 1-phosphate, one 
anomeric form of which has been made by enzyma- 
tic methods and is the intermediate in the biosyn- 
thesis and breakdown of deoxyribonucleosides. 
This exceedingly sensitive substance has now been 
synthesized by D. L. MacDonald ; by appropriate 
modification in the synthetic pathway, the other 
anomer (not, as yet, reported in biochemical sys- 
tems) was also made. 

An unusually simple method for the chemical 
preparation of aldose 1-phosphates has been de- 
vised by D. L. MacDonald, the first step being 



simply fusion of a fully acetylated aldose with 
anhydrous phosphoric acid. a-D-Glucopyranose 
and a-D-galactopyranose 1-phosphates were syn- 
thesized in the course of this research. 

In continuation of studies on the chemistry of 
ribofuranose derivatives E. K. Ness has investi- 
gated the behavior of a number of l-0-a,cjl-2-0-p- 
nitro-benzenesulfonyl-3, 5-di-(9-benzoyl-/J-D-ri- 
boses with various acyloxy ions. In each case three 
steps in a concerted reaction took place: (1) the 
acyloxy ion made an a-attack at carbon one, (2) 
the j8-acyloxy group at carbon one migrated to 
carbon two and (3) the ^-nitrobenzenesulfonyloxy 
group was ejected from carbon two with inversion 
of the configuration of that carbon. 

In the course of the above work 3,5-di-6'-ben- 
zoyl - 2 - — p - nitrobenzene - sulf onyl - ,8-D-ribosyl 
bromide was prepared. In subsequent research 
this substance was found to undergo an elimina- 
tion when treated with sodium iodide in acetone 
solution. The product, 3,5-di-6'-benzoyl-l,2-dide- 
oxy-D-eryi^Aro-pento-furanos-l-ene, is the fii'st 
known sugar glycal having a furanose ring. Its 
structure was confirmed both by conventional 
chemical means and through its nuclear magnetic 
resonance spectrum. The exceptional reactivity of 
the substance suggests its possible utility in the 
synthesis of derivatives of biochemical impor- 
tance. 

The Committee on Biological Chemistry of the 
Division of Chemistry and Chemical Technology 
of the NAS-NEC has been engaged for the last 
few years in efforts to formulate specifications for 
organic substances of biochemical importance. 
As members of the Sub-Committee on Carbohy- 
drates, H. G. Fletcher, Jr. and H. B. Wood, Jr. 
have carried out an exhaustive study of a wide 
variety of carbohydrates and evolved criteria and 
standards of purity which have now been pub- 
lished by the NAS-NEC in the hope that they will 
prove useful both to the producers and users of 
biochemicals. 

Condensation of nitromethane with the perio- 
date oxidation product from methyl a-D-glucopy- 
ranoside yields 3-nitronates principally with the 
D-manno and D-gluco configurations. Spontane- 
ous epimerization of the 3-nitronates in aqueous 
solution yields principally glycosides with the 
jy-talo and D-galacto configurations. From the 
former, crystalline 3-amino-3-deoxy-a-D-talose 



178 



ANlSrUAL REVIEW OF INTRAMURAL RESEARCH 



hydrochloride has been obtained to complete the 
series of the eight possible 3-ammo-3-deoxy-D- 
hexoses. (H. H. Baer) 

Further studies of the sugars in the avocado and 
8edum species have proved the first known nat- 
urally occurring nonulose to be D-erythro-l-^xico- 
nonulose. Several octuloses were synthesized, 
and 'D-glycero-T>-gulo-OQ,t\x\os& became the first 
crystalline representative of this series. (N. K. 
Eichtmyer and H. H. Sephton) 

Medicinal Chemistry 

The immunochemical program of the section 
(T. D. Perrine) has been continued along three 
main lines : synthetic antigens, Vi antigen and TB 
cell-walls. Copolymerization of styrene and 
^-acetoxystyrene has been successful. The re- 
sultant copolymer was coupled with a diazotized 
napthylamine disulfonic acid to yield a colored 
polymer which showed some antigenic activity 
when tested in rabbits. Eleven other test antigens 
based on polyvinyl alcohol have been prepared 
and an interesting versatile, intermediate, p-hy- 
droxyphenylpyrrolidone has been synthesized in a 
3-step sequence for possible selective N-vinylation. 
Gas chromatographic assay for acyl groups in the 
Vi antigen studies has been worked out. The 
American Instrument Company, in collaboration 
with Mr. Perrine, has produced a prototype of the 
cell-wall press developed by Mr. Perrine. 

The pharmacology unit (N. B. Eddy, consultant 
and advisor) continues to act as a clearing agency 
for all narcotic-analgesic type substances pre- 
pared in the U.S., and abroad in their complete 
evaluation for analgesic effect, toxicity and addic- 
tion liability. In fulfilling this function some 80 
new compounds have been assayed by this unit 
(Mrs. Louise Atwell assisted by Mrs. Josephine 
Goodwin) for both oral and parenteral effect in 
the mouse, while for at least 20, acute toxicities 
have been determined. On the basis of the results 
many of these compounds have been recommended 
for addiction evaluation in the monkey (Dept. of 
Pharmacology, University of Michigan). More 
promising substances have been further studied at 
the Addiction Kesearch Center, Lexington, Ken- 
tucky. Before granting a license for marketing, 
the advice of these groups is sought by the Food 
and Drug Administration. In addition, the Phar- 



macology unit has this year completed their por- 
tion of a two-year cooperative standardization 
study of the hot-plate method for assaying quan- 
titatively narcotic analgesics. All data obtained 
in these studies and in our own program, includ- 
ing acute toxicity experiments, have been subjected 
to careful probit analysis. (Mrs. Wendy Ness) 

Following completion of a 3-5 year study, data 
have been assembled which corroborate earlier pre- 
liminary findings. Doses of 20 nig./kg. ( 10 times 
the analgesic dose) of morphine repeated at in- 
tervals of 2.5-3 months over a period of 15 months 
induces a degree of tolerance to analgesic effect in 
the rat almost as great as that induced by 70-day 
addiction periods in which animals were given as 
much as 100 mg./kg. twice daily. One year after 
withdrawal some tolerance to analgesic effect still 
persists. On the other hand, recovery as judged 
by another parameter, swimming capacity, is com- 
plete at the end of six months. (J. Cochin) 

A reproducible, sensitive, rapid method of detec- 
tion and identification of narcotic analgesics and 
metabolites in the urine of patients has been de- 
veloped (J. Cochin in collaboration with J. Daly, 
Metabolites) . The method is based on thin layer 
chromatography and may prove applicable to bar- 
biturates, phenothiazines, etc. 

Eeduction of diethyl 2, 5-diketohexahydroter- 
ephthalate (I) (J. Murphy) with lithium alumi- 
num hydride has led to a variety of products from 
which 1, 4-cyclohexadiene-l, 4-dimethanol could be 
isolated in 26% yield. Catalytic reduction of I in 
acetic acid produced a-1, 4-dicarbethoxy-2, 5-di- , 
hydroxycyclohexane (all cis configuration based on 
infrared data and mode of formation) in 62% 
yield. These compounds are of interest because , I 
of their resemblance to intennediates in carbohy- ' ' 
drate metabolism and they offer possibilities of 
preparing analogs with carbocyclic rather than ij 
heterocyclic structures. ' 

Alkaline hydrolysis followed by enzymic de- 
phosphorylation of the teichoic acid present in the 
cell- awls from Lactobacillus arabinosus, gave 4-0- 
(a-D-glucopyranosyl) -D-ribitol (II) as a major 
product. The synthesis of this a-anomer has been 
achieved (L. J. Sargent) in eight steps from D- 
ribonolactone. Systematic protection by, and re- 
moval of, various functional groups was necessary. 
Furthermore, inasmuch as a mixture of the ano- 
meric glucosyl ribitols was obtamed it was ex- 



Ml 



NATIONAL ESTSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



179 



pedient to incubate this mixture with /8-glucosidase 
and remove monosaccharides. The resultant crude 
material was purified by preparative paper 
chromatography to give crystalliue II which 
proved to be identical with the cell- wall degrada- 
tion product. 

The acid cyclization of 3,4-diaIkyl 2-(^-meth- 
oxybenzyl) -1-methyl - 1-,2,5,6-tetrahydropyridines 
leads invariably to diastereoisomeric (at carbon 
9 ) 5,9-dialkyl-2'-hydroxy-2-methyl-6,7-benzomor- 
phans in yields of 70-80% for the a-isomer and 
2-8% for the /3-isomer the yield and proportion 
dependiiag on the temperature and the cyclizing 
agent. Methiodide rate-formation studies (based 
on non-aqueous titration of unreacted base) (S. E. 
FuUerton, E. L. May) complemented by NMR 
data in the dimethyl series (E. D. Becker, Lab. of 
Physical Biology) prove conclusively the valadity 
of our initial assumption that in the px'edominant 
(a) isomers, the 5,9-dialkyl radicals are in cis- 
relationship to each other, the 9-alkyl being 
oriented away from nitrogen (axial for the hydro- 
aromatic ring) m this azabicyclo system. In 
cliloroform at 25° the a-isomers are converted to 
their methiodides 10-20 times faster than the /3- 
compomids where the orientation of the 9-alkyl 
substituent is close enough to the nitrogen to 
provide steric hmdrance. This correlates the 
stereochemistry of the a-isomers with that of mor- 
phine and the morphinans at the three common 
asymmetric centers, and the analgesically more 
potent yS-isomers with the rarer but more powerful 
isomorphinans. New alkyl-2'-hydroxy-6,7-benzo- 
morphans (several of which were degraded to 
alkyl - 2-dimethylamino - 7 - methoxy - 1 ,2,3 ,4 - tetra- 
hydronaphthalenes (J. H. Ager, S. E. FuUerton, 
S. Saito) ) fomid to have diuretic properties syn- 
thesized fi'om alkylpyridines via the Stevens 
modification (E. M. Fry) or the Grewe synthesis 
and evaluated as analgesics or as tranquilizers are : 
5-ethyl-2-methyl-;5-ethyl-2-phenethyl- (S. Saito) ; 
6-propyl-2-methyl- ; a- and ;8-5,9-dipropyl-2- 
methyl (J. H. Ager) ; a- and yS-5-ethyl-2,9-di- 
methyl; a- and ;8-9-ethyl-2,5-dimethyl (S. E. 
FuUerton) . Several of the benzomoi-phans were 
also degraded to previously unknown naphthalene 
derivatives, to serve as reference compounds in 
structure elucidations. Also synthesized in eight 
steps (H. Kugita, S. Saito) from a simple ben- 
zene derivative was 5-carbethoxy-2-methyl-6,7- 



benzomorphan, in essence a hydrid of the benzo- 
morphan and meperidine classes of analgesics. 
Cyclization experiments (S. E. FuUerton, J. H. 
Ager, E. L. May) designed to improve the yields 
of the more interesting y8-5,9-diakyl compounds 
(some of which are 25 times more potent than 
morphine and will not support morpliine addic- 
tion in the monkey) have been moderately success- 
ful. An ingenious alternative 7-step synthesis 
for ;8-2'-hydroxy-2,5,9-trimethyl-6,7-benzomor- 
phan, not applicable to higher homologs, has been 
developed (S. Saito), starting material 7-meth- 
oxy-;8-tetralone. Basic research in the pyridine 
series (E. M. Fry) is being conducted with a view 
of preparing intermediates wliich might undergo 
ready cyclization to produce practicable amounts 
of the above-mentioned y8-isomers. The decompo- 
sition of the hydrochloride salt of 2-benzyl-6- 
cyano - 1,3,4 - trimethyl - 1,2,5,6 - tetrahydropyridine 
gives as a major product, a basic compound 
(a 303 mu) which, based on analogy with simpler 
systems, could be the desired 2-benzyl-l,3,4-tri- 
methyl-l,2,3,6-tetrahydropyridine with the 3- 
methyl and 2-benzyl groups in ^raTW- juxtaposition. 

Metabolites 

A review, as well as preview, of the rapidly 
expanding program on "Non-Enzymatic Methods 
for the Preferential and Selective Cleavages and 
Modifications of Proteins" has been completed for 
Volume 16 of Advances in Protein Chemistry. 
A useful dichotomy of the methods involved has 
been introduced and defined by the terms "prefer- 
ential" and "selective," the former involving com- 
petitive and hydrolytic conditions, the latter non- 
competitive, non-hydrolytic and oxidative 
cleavage. Selective cleavage so far has been 
realized with peptide bonds following tryptophan, 
tyrosine, methionine, and to some extent histidine, 
as- well as y-aminobutyryl, y-glutamyl and 8- 
amino-adipyl peptides. (B. Witkop) 

The selective cleavage of y-aminobutyrylglycine 
with nitrous acid, which has been elaborated by 
J. E. Francis, has now found a practical industrial 
application in the conversion of the novel and 
important cephalosporin C to 7-aminocephalospo- 
ranic acid. 

The nonenzymatic selective cleavage of bovine 
pancreatic ribonuclease in the hands of E. Gross 



643351 — 62 



-13 



180 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



has led to a major revision of the primary sequence 
of this enzyme. In accordance with these findings 
the group at the Kockefeller Institute, as well as 
Anfinsen's group in the Heart Institute, formulate 
the crucial, and probably labile, sequence 11-18 as 
follows: Gn (11)— His (12)— Met (13)— Asp 
(14)— Ser (15)— Ser (16)— Thr (17)— Ser (18). 
A major methodological improvement has been the 
fractionation of cyanogen bromide-treated ribonu- 
clease on a column of Sephadex G-50. Tlus pro- 
cedure permits the clean separation of the "chemi- 
cal tail peptide" (ribonuclease positions 1-13) and 
of homoserine (lactone) from the cleavage of Met 
(29) — Met (30) from the core of ribonuclease. 

The new fractionation on Sephadex has made it 
possible to prepare the so-called S-peptide obtain- 
able from ribonuclease by the action of subtilisin 
in a dependable manner in liigh yield. The prep- 
aration of this S-peptide has been a major problem 
in the laboratories of Richards at Yale and An- 
finsen in the Heart Institute. The availability of 
S-peptide has made it possible for E. Gross to 
study the nonenzymatic cleavage with cyanogen 
bromide at the peptide bond following Met (13). 
The cleavage products, a tridecapeptide and a 
heptapeptide, have been purified, characterized 
and analyzed. The new preparation method for 
S-peptide foregoes the use of trichloroacetic acid. 
It will now be possible to study the suspected labil- 
ity of the S-peptide under acid conditions in detail 
and to explore the possibility of rearrangements or 
even sequence tautomerism. 

C. M. Foltz has studied the influence of alkyl 
groups on the cleavage of sulf onium salts derived 
from ethyl N-acetylmethionylglycinate. The best 
percentage yields in peptide cleavage were ob- 
tained with iodoacetamide as alkylating agent. 
The course of the alkylation at 35-40° was fol- 
lowed by argentometric titration. A comparison 
of yields in the cleavage of iodates, acetates and 
nitrates of methioninesulfonium peptides showed 
that the cleavage yield was independent of the na- 
ture of the anion. 

By means of N-bromosuccinimide oxidation, S- 
carboxymethyl ribonuclease has been cleaved se- 
lectively next to the 6 tyrosine C-peptide bonds to 
release 6 new NHa-terminal amino acids. The re- 
sults obtained by L. A. Cohen and J. G. Wilson are 
in full accord with the sequence proposed by in- 
vestigators at NHI and at the Rockefeller 
Institute. 



The program on enzyme fragmentation lias been 
started by C. M. Foltz with pepsm, which has been 
cleaved by nonenzymatic methods under condi- 
tions which did not lead to denaturation but to 
fractions retaining 60-80% of the original activity 
corrected by control runs. The major problem, 
which has been surmounted, is the preparation of 
pure samples of pepsin without concomitant autol- 
ysis. The aim is the preparation of an enzyme 
fragment wliich is still fully active without neces- 
sarily being fully specific, and which is of a size 
small enough for study and synthesis of the active 
center. (A. Arens) 

The antibiotic gramicidin A, homogeneous by 
the criteria of 1,000-transfer countercurrent dis- 
tribution, has been resolved by S. Ishii into several 
components of the use of analytical and prepara- 
tive thin layer chromatography. The presence of 
isoleucine in gramicidin A has been observed for 
the first time. Tlie successful structural elucida- 
tion of gramicidin A is awaiting the preparation 
of sufficient amounts of these new congeners. 

The mtramolecular participation of amide 
groups in peptides of unsaturated amino acids has 
been studied in some detail by N. Izumiya and 
A. V. Robertson. Amides and peptides of N- 
acylated DL-allylglycine or DL-methallylglycine 
reacted in aqueous buffer systems or water with N- 
bromosuccinimide with liberation of ammonia or 
the peptide component. The liberation of ammo- 
nia, glycine, or glycinamide was a linear fimction 
of the added reagent and after the addition of 1-2 
moles of N-bromosuccinimide 60-80% of the amine 
component was liberated, as measured by ninhy- 
drin assay. On a preparative scale the primary 
amides gave products different fi'om the lactones 
obtained from the allylglycine peptides. Their 
insolubility in water, solubility in nonpolar sol- 
vents, analytical data, and content of one covalent I 
and one active positive bromine led to their formu- 
lation as N",C-dibromoiminolactones. Unsaturated 
amino acids which contain the double bond in a 6- 
or 5-membered ring participated only in the release 
of ammonia, but not of, e.g., glycine, from the re- 
spective precursors. 

In collaboration with N. Izumiya, a stereospe- 
cific approach to the synthesis of aZZo-hydroxy-L- 
and -DL-proline and threo-\\yAvo:iy-lj- and -DL- 
ornithine has been worked out based on the reac- 
tion of N-carbobenzyloxyallyl-L- and -DL-glycine 
with N-bromosuccinimide. 






NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



181 



I Professor Izumiya is continuing this approach 
at the Laboratory of Biochemistry at Kyushu Uni- 

. versity in order to make accessible the racemic and 

( optically active forms of hydroxylysine and allo- 
hydroxylysine. The new synthetic route will be 
utilized for the preparation of L-hydroxyarginine 

' and L-aZZo-hydroxyarginine and relative rates of 
enzymatic deguanidination by arginase, and fur- 

; thermore for the synthesis of homoarginine, 
arginine and lysine peptides containing hydroxy 
groups for the study of their substrate specificity 
with trypsin and papain. 

Detailed nuclear magnetic resonance studies by 
A. V. Robertson in collaboration with E. Becker 
have confirmed the structure of the oxidation prod- 
uct of N-carbobenzyloxydehydroproline amide to 
be the unusual dicarbinolamide, formally the con- 
densation product of benzylcarbamate with maleic 
dialdehyde. In a cooperative study Professor G. 
Fodor of the Hungarian Academy of Science in 
Budapest is testing this new dicarbinolamide asi a 
partner in the Mannich reaction wliich should lead 
into the difficulty accessible tropinone series. 

J. E. Francis and A. V. Eobertson succeeded in 
preparing the two isomeric betaines derived from 
dehydroproline differing only in the position of 
the double bond. The unstable 3,4-dehydro-DL- 
stachydrme rearranged easily to the isomeric 
optically inactive betaine which was found to be 
identical with the water elimination product from 
the two diastereoisomeric 3-hydroxy-stacliydrines 
from Courbonia virgata. 

3-Hydroxyproline, a novel hydroxyamino acid 
occurring as a regular constituent in collagen to 
the extent of 1% of 4-hydroxyproline, has been 
synthesized by A. V. Eobertson from 3,4- 
dehydroproline by the hydroboration reaction. A. 
Robertson, now professor at the University of 
Sydney, is carrying on this research by trying to 
prepare the cis- and #ra7W-diastereoisomeric DL- 
and L-3-hydroxyprolines. 

Poly-3,4-dehydro-L-proline has been prepared 
by two different routes. Like poly-L-proline the 
new polypeptide has two metastable interconver- 
tible forms which have been characterized by their 
rotational values, supplemented by rotatory dis- 
persion and infrared studies. This investigation 
has been a joint one with Dr. Arieh Berger from 
the Weizmann Institute of Science. 



Selectively 4-tritiated natural hydroxy-L-pro- 
line has been synthesized by A. V. Robertson via 
4-keto-L-proline by reduction with ]SraBT4. A 
novel procedure for the separation of diastereoiso- 
meric hydroxyprolines, developed by S. N. Bim- 
baum, NCI, has been utilized to advantage, em- 
ploymg controlled buffer systems and ion ex- 
change columns. E. Katz, jointly working in the 
Laboratory of Clinical Biochemistry, NHI, and 
LC, has made a detailed study of the incorporation 
of selectively labeled hydroxyproline and proline 
into the peptide part of the various actinomycins 
elaborated by Streptomyces antibioticus. 

A comprehensive synthetic program on 3- or 
4-fluoro-, 3,4-dihydroxy-, and 3,4-epoxyprolines 
and the respective halohydrins has been started 
by A. V. Eobertson. These compounds are being 
supplied to the laboratories of S. Udenf riend and 
A. Sjoerdsma, where screening tests are being de- 
veloped for compoimds specifically inhibitory to 
the incorporation of (hydroxy) proline into col- 
lagen. 

A novel tricyclic metabolite of serotonin, has 
been discovered in dehydrobufotenine, a major 
indole component from the South American toad 
{Bufo marinus) . A new isolation procedure util- 
izing ion exchange columns has made possible the 
easy preparation of dehydrobufotenine from ex- 
cised toad parotid glands. In the elucidation of 
its structure some new chemical reactions have 
been helpful, but a complete and detailed analysis 
of the nuclear magnetic resonance spectrum of de- 
hydrobufotenine, compared with bufotenine, fur- 
nished rigorous proof for the proposed structure, 
whose biosynthesis is under investigation. The 
synthesis is in progress in the laboratory of Pro- 
fessor a Burgstahler, Univ. of Kansas, who 
started this project as an NIH visitor during the 
summer of 1961. 

In addition to the biosynthesis of dehydrobufo- 
tenine in toad parotid glands, F. Marki, with 
H. Weissbach, has started an investigation of the 
biosynthesis of adrenoglomenilotropin in beef 
pineal gland. A highly sensitive assay for this 
interesting tetrahydroharman derivative has been 
worked out. Evidence has been secured for the 
occurrence of other tetrahydroharman derivatives 
occurring in the glands. 

In collaboration with J. Axelrod, F. Marki has 
completed a detailed study of the O- and 



182 



ANNUAL REVIEW OF INTRAMUBAL RESEARCH 



N-methyltransferases occurring in the parotid 
glands of Bufo mannus. In the course of this in- 
vestigation he discovered epinine for the first time 
in the animal kingdom. 

Jolxn Daly, in collaboration with the Laboratory 
of Clinical Biochemisti-y, has made a careful study 
of the mechanism of action, assay method and sub- 
strate specificity of the enzyme that converts dopa- 
mine to norepinephrine. These studies have made 
use of substrates labeled with C^*, tritium and O^®. 
In collaboration with Dr. David Samuels of the 
Weizmann Institute, who is holder of a special 
NIH grant, methods for the micro assay of O^^ 
are being worked out. 

John Daly, in collaboration with Professor L. 
Horner of the Univ. of Mainz, has shown that the 
addition of methanol to quinones of dopamine 
obeys the 1,2-principIe followed by allylic rear- 
rangement. Using nonenzymatic and enzymatic 
methods all three isomers of the monomethyl 
ethers derived from 2,4,5-trihydroxyphenethyl- 
amine have been synthesized. 2,4-Dihydroxy-5- 
methoxyphenethyl amine has been discovered as a 
new significant metabolite of dopamine in rats. 
The metabolic fate of mescaline in vivo and 
vitro has been further studied by John Daly, who 
discovered mescalol as a new hydroxy derivative 
resulting from the action of norepinephrine syn- 
thetase on mescaline, which is 3-5% as good a 
substrate as dopamine. 

Y. Kanaoka completed the difficult synthesis of 
6-hydroxynnorepinephrine, which was found to be 
a substrate for 0-methyltransf erase, yielding 2,4- 
dihydroxy-5-methoxyphenethanolamine, which is 
not formed from 2,4-dihydroxy-5-methoxyplien- 
ethylamine by the action of norepinephrine syn- 
thetase. The possible confluence of the metabolism 
of mescaline with the metabolism of dopamine is 
under investigation. 

In collaboration with J. Cochin, John Daly has 
applied thin layer chromatography to the separa- 
tion of alkaloids, barbiturates, phenothiazine and 
related compounds and their identification as such 
or as metabolites in urine. The method is unusu- 
ally sensitive and promises to be of forensic value. 
B. Witkop, while Visiting Professor at the Univ. 
of Kyoto, collaborated with S. Senoh, Institute of 
Food Chemistry, Osaka, Japan, on the compara- 
tive role of metal cations in enzymatic and non- 
enzymatic 0-methylation reactions. The non- 



enzymatic and enzymatic mono-0-methylations 
of certain catechol derivatives produce mixtures 
of isomeric mono-0-methyl ethers in a ratio de- 
pendent on pH and on the type of added bivalent 
metal cation. In enzymatic 0-methylations with 
0-methyltransf erase the total yield of 0-methyla- 
tion products as a function of metal ions decreases 
in the following order: Mg+* > Zn++ > Mn** > ? 
Co++ > Ni+* > Fe*+ > Cu++. The effectiveness of 
bivalent cations in promoting nonenzymatic 
m-0-methylations witli dimethyl sulfate increases 
in the following order : Mg++ < Zn+* < Mn*-' < 
Co*-" < Ni++ < Fe-"* < Cu++. These observations 
are discussed in terms of the 2:1 catechol-metal 
complex and the enzyme-metal bridge complex 
operative in tlie enzymatic and nonenzymatic O- 
methylation reactions. 

In collaboration with S. Udenfriend and J. 
Pisano, L. A. Cohen has synthesized several di- 
peptides of y-aminobutyric acid which have been 
identified with natural peptides occurring in brain |j 
tissue. The significance of such peptides in neuro- 
chemistry is currently under investigation. 

In collaboration with G. Glenner, L. A. Cohen 
and W. M. Jones have prepared a variety of histo- 
chemical substrates designed to locate new proteo- 
lytic enzymes in specialized tissues. To date, the 
study has demonstrated a trypsin-like enzyme 
which is not inhibited in the same manner as 
trypsin. Of particular interest is the fact that 
the enzyme shows considerably higher activity in 
some carcinoid tissues than in normal. 

In an attempt to elucidate the role of Coenzyme 
Q in oxidative phosphorylation, W. Diircklieimer 
and L. A. Cohen have imdertaken the preparation 
of a new class of labile compounds, cyclohexadi- 
enone phosphates. Oxidation of a-tocopherol 
with N"-bromosuccinimide and acetate ion has 
yielded a cyclohexadienone acetate, an analog of 
the desired dienone phosphate. 

A detailed study of the reaction of ninhydrin 
with various classes of amines and amino acids has 
suggested that the generally accepted mechanism 
for the reaction may require modification. On the 
basis of new information, L. A. Cohen and W. M. 
Jones hope to apply the ninhydrin method to the 
quantitative assay of various phenolic amines pres- 
ent in physiological systems. 

In collaboration with E. Mosettig and J. Steele, 
L. A. Cohen has applied nuclear magiietic reso- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



183 



nance spectroscopy to the elucidation of structure 
and sterochemistry in rearrangement products of 
dehydroergosterol. The findings have provided a 
considerably simplified method for the stereochem- 
ical analysis of ring B ai'omatic steroids. 

Steroids 

Steviol-Isosteviol 

The inversion with butyl lithium at carbon atoms 
8,13 of the "nor keto acid" (from steviol) to an 
"iso nor keto acid" and the Cotton effects of these 
two acids confirm our previous conclusion that in 
steviol-isosteviol the 9-H is ^-oriented. The iden- 
tity of stevane A with (-) a-dehydrokaurene (of 
Briggs et al.) established the absolute configura- 
tion of centers C9, Cs and C13 in the latter. The 
identity of stevane B with the degradation hydro- 
carbon from garryfoline (Djerassi et al.) confirms 
the absolute configuration of these centers in the 
garrya alkaloids, and therefore also in the alka- 
loids of the atisine group. 

A number of physical measurements of ap- 
propriate and comparable degradation products 
from garryfoline, atisine and steviol-isosteviol con- 
firm the tentative assimiption (by Edwards et al.) 
of the "wrong" A/B juncture in these three groups. 
Thus the absolute configuration of all asymmetric 
centers (5-7) m the above groups and the kaurenes 
is established. This ia turn brings to conclusion 
the combined efforts (startmg about 1953) of 
Wiesner (Fredericton, N".B.), Edwards (Ottawa), 
Pelletier (New York), Djerassi (Stanford), 
Briggs (Auckland) and our laboratory. (E. 
Mosettig, U. Beglinger and J. A. Waters) 

The (7i7, Cio-Isomers of Cholestane 

A^-Cholestane-3, 16, 27-triol and the corre- 
sponding 5, 6-dihydro compound, give on tosyla- 
tion and detosylation, among others, A^-cholesten- 
16j8-ol and the corresponding dihydo derivative 
respectively. On dehydration of the latter A°,^^- 
cholestadiene and A^'^-cholestane were obtained m 
pure form {ca. 35%). The corresponding A"- 
isomers were formed but could not be isolated in 
ci-ystalline form. The locations of the double 
bonds were established by N.M.E. spectroscopy. 
The reduction of the A^^-enes, in conj miction with 
previous work (20-iso-A^*'-cholestane), allowed as- 
signing the definite structures 20-iso cholestane 



and 17- and 20-isocholestane respectively to the 
reduction products. These compounds are of 
practical and theoretical interest m view of stereo- 
specific biosynthetic pathways of natural sterols 
and tetracyclic triterpenes isomeric at the C17-, 
C2o-centers. (G. Nair and E. Mosettig) 

Microbiological Hydrowylations of Steroid Al- 
kaloids and Steroid Sapogfenins 

By employing Helicostylum piHforme and a 
medium consisting of peptone, cornsteep liquor, 
dextrose and tap water it has been possible to hy- 
droxylate solasodine and tomatidine. The reac- 
tion products obtained are respectively: lla-hy- 
droxy- and 7y8-hydroxysolasodine, 9a-hydroxy-, 
7a-hydroxy- and 7a,lla-dihydroxy tomatidine. 
By the same means diosgenin was converted to 
7y8,lla-dihydroxy- and lla-hydroxy-7-oxo-dios- 
genin. These hydroxylated derivatives may serve 
as starting materials for hormonal steroid analogs 
not accessible by conventional diemical proce- 
dures. Theoretically significant is the decisive 
effect of the configurations of the C25 asymmetric 
center upon the course of microbiological hydroxy- 
lation. (Y. Sato and S.Hayakawa) 

Biogenesis in the Slime Mold: The slhne mold 
Dictyostelium discoideum incorporates C^*-acetate 
or C^*-mevalonate into A^^-stigmasten-3/?-ol. 
Studies on this incorporation during the various 
stages of the life cycle revealed that it occurred 
selectively in the vegetative stage. These findings 
are significant since this stage precedes the aggre- 
gation of the single cells into a pseudoplasmodium, 
and A^^-stigmasten-3^-ol has been shown to be 
involved in this phenomenon. An unknown radio- 
active compound was found to be attributable to 
E. coli used in the growth mediimi. Chromato- 
graphic evidence suggests that this material may 
be a sterol. (D. F. Joluison and E. Heftmann) 

Biogenesis of Plant Steroids: Mevalonic acid 
was incorporated into stigmasterol isolated from 
tomato fruits, while sodium acetate was not. The 
reverse pattern was rej)orted earlier in Dioscorea 
tuber homogenates. Stem feeding of young D. 
sjncidiflora plants with radioactive mevalonic acid 
resulted in a high incorporation into steroidal 
sapogenins and /^-sitosterol, the latter havmg a 
very high specific activity. Degradation work is 
in progress to establish the pattern of labelmg, and 
the biosynthetic relationships between the sapo- 



184 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



genins and j8-sitosterol. (E. D. Bennett and E. 
Heftmann) 

Steriod Sulfur Analogs: Two new approaches 
to the synthesis of 11-thiol steroid analogs failed : 
(1) The hydroboration of A°'"-steroids and sub- 
sequent treatment with hydrogen polysuliide did 
not introduce the SH group. (2) Microbiological 
hydroxylation of appropriate 19-norsteroids with 
a variety of fungi (which introduce the 11-hy- 
droxyl group in normal steroids) gave only mini- 
mal amounts of 11-hydroxy compounds wliile the 
main products proved to be 10-hydroxylated de- 
rivatives. This closes an important avenue to 
A"' '"'-19-norsteroids. It is assumed that the 
absence of the methyl group at Cio enhances the 
reactivity of the 9,11-double bond or the cor- 
responding epoxy ring, and thus allows the intro- 
duction of an S-group at position 11. (Y. Ueda 
and E. Mosettig) 

Steroid Anlyzer: The gradient system of the 
automatic steroid anaylzer developed in our lab- 
oratory, permits programing any desired solvent 
ratio for gradient elution chromatography of ster- 
oids in a mixture. Preliminary studiles with sev- 
eral different gradients indicate that this feature 
will facilitate the separation and quantitative esti- 
mation of closely related steroids in a mixture. 
Experiments are continuing on the effects of these 
gradient changes on chromatographic separations. 
(D. F. Johnson, E. Heftmann) 

Sfectrosccpic and SfectrofolarimetriG Studies 

(a) The decision between A^'^ and A^'^ in 
arborine could not be made on the basis of chem- 
ical results. U. V. and I. E. spectra were equally 
inconclusive. Finally, N.M.E. spectra established 
the structure of arborine as l-methyl-2-benzyl-l,4- 
dihydroquinazol-i-one. (H. K. Miller, L. A. 
Cohen and E. N. Chakravarti) . (b) 784 infrared 
spectra have been made in support of investiga- 
tions in this and other laboratories, (c) An in- 
frared analytical procedure for the evaluation of 
cis-trans olefinic fatty acid compensation in mate- 
rials submitteed is under design, (d) The pre- 
cision of the Eudolph spectropolarimeter wais 
greatly increased by stabilization of the high pres- 
sure xenon arc lamp, replacement of the polarizing 
and analyzing prisms, replacement of the high 
voltage power supply and the installation of a 
magnetic commutator system. (H. K. Miller and 
Mrs. A. W. Wright) 



Microanalytical Services 

Approximately 8,000 analytical determinations 
were carried out for 140 of the NIH research staff 
and for several scientists in other Government 
agencies. These included 7,800 routine micro- 
analyses and approximately 225 non-routine anal- 
yses requii'ing varying degrees of laboratory and 
literature investigation. 



I 



\\ 



LABORATORY OF MOLECULAR BIOLOGY 

The preparations for occupancy, in Building 2, 
of the newly organized Laboratory of Molecular 
Biology are approaching completion. Four sec- 
tions will be assembled. These will be : 

Section on Metabolic Enztmes. This section 
is the Laboratory of Biochemistry and Metabolism 
(q.v.). This section is headed by Dr. Gordon 
Tomkins and includes Drs. Miles, Nirenberg and 
Maxwell, together with research fellows and sup- 
porting staff. 

Section on Chemical Genetics. This section 
is headed by Dr. Harvey Itano, presently in the 
Laboratory of Experimental Pathology (q.v.) 
Dr. W. J. Dreyer, presently of NHI, will join this 
Section. 

Section on Physical Chemistry. Tins group 
is headed by Dr. Gary Felsenfeld, who has been 
brought to NIAMD from the University of Pitts- 
burgh. Tlie studies carried out in the Section on 
Physical Chemistry will primarily be concerned 
with the relationship between structure and func- 
tion in nucleic acids and proteins. Major projects 
whicli are now being established deal with the role 
of the negatively charged backbone of nucleic 
acids in the stabilization of ordered structures, the 
kinetics of DNA denaturation, the structure of the 
active site in copper proteins (Felsenfeld, 
Sandeen, Smith) the kinetics of polynucleotide 
interaction and the transport behavior of poly- 
electrolytes (Eoss). Two studies presently in 
progress (Felsenfeld, in collaboration with P. Von 
Hippel, Dartmouth Medical School) are concemed 
with the dependence of the apparent denaturation 
temperature upon the wave length used to detect 
the loss of ordered structure of nucleic acids and 
with the specificity of deoxyribonuclease for vari- 
ous structural forms of deoxvribonucleic acid. 



NATIONAL INSTITUTE OP ARTHRITIS AND METABOLIC DISEASES 



185 



Section" on Molectjlak Steuctuee. This Sec- 
tion is headed by Dr. David E. Davies, formerly of 
NIMH. The members of the Section on Molecular 
Structure (Davies, Gellert, Sigler and Skinner) 
have been concerned during the past year with 
structural studies on nucleic acids and proteins 
using x-ray diffraction methods. In the area of 
nucleic acid structure a variety of investigations 
has been undertaken concerning structural aspects 
of both natural and synthetic nucleic acids. A 
systematic study of the synthetic polydeoxyribo- 
nucleotides lias been commenced with a demonstra- 
tion of the structural similarity of the synthetic 
polymers containing adenine plus thymine, and 
adenine plus 5-bromouracil with natural DNA. 
The mode of bindmg of the mutagenic acridine- 
class of dyes to DNA has been investigated with 
results which rule out some present hypotheses 
concerning the mechanism of the mutagenic action 
of these dyes. The organization of DNA, in the 
T4 bacteriophage has been studied by flow bire- 
fringence methods, and it has been shown that only 
part of the DNA is perferentially oriented paral- 
lel to the long axis of the phage. 

In the area of protein structure attention has 
been f ocussed on the proteolytic enzymes. Pre- 
liminary crystallographic investigations have been 
carried out on several of these and a systematic 
search for isomorphous heavy atom substitutions 
in y-chymotrypsin is now in progress. 



LABORATORY OF NUTRITION AND 
ENDOCRINOLOGY 

Folic Acid 

The intact rat, deficient in vitamin B12, is unable 
to metabolize Carbon-2 (ring) of histidine and 
consequently excretes formiminoglutamate. Im- 
mediately following the injection of methionine, 
the rat acquires the ability to metabolize Carbon-2. 
Examination of the folate distribution patterns of 
the livers of such rats has shown (1) that N^- 
methyltetrahydrofolate accumulates in the liver 
of the B12 deficient rat and (2) after methionine 
administration, the methylfolate concentration is 
greatly reduced and the liver now contains large 
stores of N^^-f ormyltetrahydrof olate. It thus ap- 
pears that methionine provides an acceptor for the 



methyl present in the folate, freeing the latter for 
the metabolism of Carbon-2 of histidine. It is 
concluded that in vitamin B12 deficiency, folic acid 
is "trapped" in the tissues as methyl folates and 
functional folic acid is released only after a suita- 
ble acceptor for the methyl group is provided. 

Pref olic A has been identified as methyltetrahy- 
drof olic acid with the methyl group most prob- 
ably in the 5-position. On enzymatic oxidation 
pref olic A was converted to 5, 10-methylenetetra- 
hydrofolic acid which disassociates to yield form- 
aldehyde and tetrahydrofolic acid. Menadione 
(vitamin K3) functioned as the hydrogen acceptor. 
Prefolic A was enzymatically synthesized using 
reduced di- or triphosphopyridine nucleotide as 
the hydrogen donor in reducing 5, 10-methylene- 
tetrahydrof olic acid. Prefolic A was synthesized 
chemically by reduction of the 5, 10-methylenetet- 
rahydrof olic acid with sodium borohydride. 

The gas gland of the Portuguese Man of War 
{Physalis fhysalia) excretes CO into its float. 
Serine appears to be the primary source of the CO. 
The gland responsible for its secretion contains a 
high concentration of folic acid (Wittenberg, 
1960). In collaboration with Wittenberg (Albert 
Einstein College of Medicine, N.Y.) the gas gland 
has been examined for its folate pattern. The 
results indicate the folates consist essentially of 
N^°-formyl derivatives of tetrahydrofolic acid, 
diglutamyl-tetrahydrofolic acid, and as yet, an 
uncharacterized polyglutamyl derivative of folic 
acid. No methyl derivatives are detectable. 
These observations are consistent with the view 
that the CO formed by the gas gland originates 
from the B carbon of serine by way of an 
N^^-formyltetrahydrofolate derivative. 

Purification of the enzyme from chicken liver 
catalyzing the reduction of N^'-formylfolic acid 
to the tetrahydro-level has led to the conclusion 
that this enzyme is probably identical to folic re- 
ductase. However, the observation that tliis re- 
duction is stimulated by the presence of folic acid 
itself has suggested a second reaction occurring 
in the absence of TPNH : that N^^-f ormyldihydro- 
folate, produced as an intemiediate product via 
TPNH, is reduced to the tetrahydro-level by 
tetrahydrofolic acid. Preliminary observations 
indicate the reaction occurs at a rate much higher 
than the reduction of N^^-formylfolic acid by 
TPNH. 



186 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Vitamin B12 

The vitamin B12 deficient cliick excretes for- 
miminoglutamic acid (FGA) when given a sup- 
plemental dose of histidine only if the diet con- 
tains a borderline level of methionine. That 
methionine is not all-important in determining the 
excretion of FGA is shown by the observation that 
chicks deficient in methionine under similar con- 
ditions do not excrete the compound unless they 
are simultaneously deficient in vitamin Bjo. These 
observations may explain the contradictory re- 
ports in the literature concerning the excretion of 
FGA by pernicious anemia patients. The varia- 
tions in the methionine content of the diets of these 
patients are probably associated with differences 
in FGA excretions. Chicks deficient in folic acid 
excrete large amounts of FGA and the rate is little 
affected by the methionine content of the diet. 
Patients deficient in folic acid also excrete large 
amounts of FGA. 

Chicks deficient in vitamin B12 and receiving a 
minimal intake of methionine show only a slight 
reduction in FGA excretion when fed homocys- 
tine (or homocysteine thiolactone-HCl) and a 
source of labile methyl groups (choline, betaine) . 
Methionine produces a marked reduction in FGA 
excretion during the first day of supplementation. 
These and other studies suggest that vitamin B12 
facilitates the transfer of methyl groups from cho- 
line or betaine to homocystine. 

Dietary Liver Necrosis 

Studies of the respiratory decline in liver homo- 
genates of rats with dietary liver necrosis indicate 
that a variety of compounds, tocopherol metabo- 
lites, menadione and short-chain coenzyme Q com- 
pounds, are protective. Although some anti- 
oxidants were very potent, lipid peroxidation was 
not involved in the respiratory decline since 
peroxidation can be prevented by levels of the 
compounds which do not alter the rate of peroxide 
formation as shown by the thiobarbituric acid 
reaction. Further support for this comes from 
the fiinding that some compounds prevent peroxi- 
dation but have no effect on the respiratory decline 
reaction. 

Previous work showed a release of deleterious 
trace elements from deficient microsomes as a 
possible cause of the respiratory decline. Com- 



bination of microsomes and mitochondria pro- 
duced respiratory decline which was prevented by 
EDTA, GSH and dimercaptopropanol. 

Factor 3-active selenium compounds were in- 
effective in preventing respiratory decline when 
added in vitro to the homogenate or to liver slices. 
However, when supiDlied to the animal either in 
the diet or by injection, they were effective in pre- 
venting respiratory decline in liver slices. In the 
homogenized liver, no such effect was seen. A 
comparison of the potency of various selenium 
compounds is under way, together with a dose and 
time exposure study. The difference between liver 
slices and the homogenate system indicates that 
the effect of Factor 3 selenium is either located 
at the cell membrane, or that it is mediated by 
an active co-factor which is diluted out during 
homogenization. Both possibilities are under 
investigation. 

Factor S-Selenium Compounds ^H 

When various synthetic selenium carboxjdic 
acids were assayed for Factor 3 activity, it was 
observed that the activity increased as selenium 
was shifted from the alpha to the beta to the 
gamma or delta position. Phenyl derivatives 
were inactive while benzyl substitution increased 
activity. Bromine substitution in the para posi 
tion of the benzene rings increased potency where- 
as methyl substitution decreased it. 

The injection of radioselenimn into rats fed 
Torula yeast diets resulted in the urinary excre- 
tion of about 30% of the radioactivity within the 
first two weeks. Chromatography and radio- 1 
autography showed the presence of five different 
organic selenium compomids in the urine. One 
of these, representing 3% of the radioactivity, 
appears to be a high molecular weight compound ; 
another minor component is a compound that is 
imstable to storage. A major component after 
chromatographic concentration is stable to evapo- 1 
ration and H2O2 treatment but is destroyed wlieul 
autoclaved in a 0.1 N NaOH solution. It has 
been isolated in radioautographically pui'e form. 
The major radioselenium compound excreted by 
the rat contains 42.5% of the radioactivity. At 
tempts to concentrate this compound have resulted 
in large losses of radioactivity. The properties 
of the compound resemble those of a-Factor 3 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



187 



whereas the other major constituent resembles fi- 
Factor 3. These compounds are being used as 
"markers" in attempts to isolate large amounts of 
the metabolites. 

The observation that sulfur amino acids have 
a strong potentiating effect on a-tocopherol led to 
a study of the action of other amino acids. Tryp- 
tophan at a dietary level of 0.2% was the only 
amino acid which showed a significant protective 
effect. Methionine and tryptophan are synergistic 
with a combination of these two amino acids per- 
mitting the rats to survive 2 to 3 times as long as 
normal on the Torula yeast diet without develop- 
ing liver necrosis. Studies are tmder way to de- 
termine the mode of action of the amino acids. 

Vitamin E Antioxidants and Selenium 

Vitamin E can be replaced in some species by 
certain antioxidants while in other species, these 
compounds appear to have only slight vitamin 
E-like activity. An explanation for this vari- 
ability comes from the observation made in this 
laboratory that there is a direct correlation be- 
tween the absorption of the antioxidants by the 
cells of the body and their efEicacy in preventing 
vitamm E deficiency symptoms. For the chick, 
low levels of l,2-dihydro-6-ethoxy-2, 2,4-tri- 
methylquinoline (ethoxyquin) and ]Sr,N'-di- 
phenyl-p-phenylenediamine (DPPD) added to a 
vitamin E and selenium deficient diet completely 
prevented all deficiency symptoms. Antioxidants 
such as nordihydrogTiaiaretic acid (NDGA) and 
di-tertiary amylhydroquinone (DAH) were inac- 
tive even when fed at high levels m the diet (0.5% ) 
white butylated hydroxy toluene (BHT) and di- 
tertiarybutyl-4-methylphenol (DBPC) showed 
intermediate activity. The inactive compounds 
were shown to be unavailable to the tissues and 
conversely, the effective compounds exerted a sig- 
nificant antioxidant action in the tissues as 
measured by in vitro lipid peroxidation tests. 

The antioxidant-like action of tissues which re- 
sults from feeding trace amounts of selenium is 
associated with the tissue proteins and is not due 
to any changes in the lipid components. Dietary 
selenium was found effective in significantly re- 
ducing the in vitro hemolysis of erythrocytes from 
vitamin E-deficient rats (another manifestation 
of antioxidant deficiency). 

643351—62 14 



Other Fat Soluble Vitamin Studies 

Vitamin A acid in the rat has been shown by 
others to replace vitamin A alcohol for growth 
functions but not for visual and reproductive 
processes. In the chick it has been found that 
vitamin A acid is slightly less active than the 
alcohol in promoting growth and preventing 
neurological disorders. High doses of the acid 
(50 to 100 /xg daily), however, did not prevent 
infection of the conjunctiva in otherwise normal 
birds. Toxic doses (2 mg daily) produced ex- 
cessive secretion of the tear glands, fissures in the 
corners of the mouth, poor growth and an un- 
thrifty appearance. 

There have been three possibilities considered 
for the origin of the benzoquinone moiety of 
ubiquinone (coenzymeQ) in animals: (a) dietary, 
(b) intestinal flora, (c) endogenous. To resolve 
this problem, a chemically defined diet devoid of 
ubiquinone was fed to young germf ree rats. They 
were found to accumulate ubiquinone in their 
livers at a rate comparable to that of conventional 
rats. These results show that the tissues can syn- 
thesize the entire ubiquinone molecule de novo and 
no exogenous source of the benzoquinone ring is 
required. 

A screening program of patients with malab- 
sorption syndrome with respect to abnormalities 
in the metabolism of vitamins A and E and caro- 
tene and ubiquinone, has turned up two patients 
with a complete absence of serum vitamin E. Tests 
to date on one patient indicate that certain symp- 
toms are responding to therapy with a-tocopherol. 

Plasmalogens 

Plasmalogens are phospholipids which contain 
a vinyl ether group. A specific procedure has been 
perfected for analyzing quantitatively as little as 
0.02 /tmole of plasmalogen. This amoimt of plas- 
malogen is present in the small size of samples 
required for enzymatic studies involving the 
metabolism of plasmalogens. 

Protein 

Studies have been carried out on the response 
of various liver components of the adult rat to 
prolonged protein deficiency followed by repletion. 
Variables studied in this investigation may be 



188 



ANTSrUAL REVIEW OF INTRAMXJRAL RESEARCH 



separated into two general classes: (1) gross and 
structural changes in the liver and liver cells and 
(2) functional changes in the liver cells as repre- 
sented by the individual enzyme components of 
the succinic oxidase system which was used as a 
model of an important oxidative system. 

Wliile the components of the electron transport 
chain are considered by many investigators to be 
spacially arranged in the mitochondrion in one 
particle, each of the individual components re- 
sponds entirely differently to a protein deficiency. 
Recently others have shown that the half-life of 
the liver mitochondrion of the rat is about 10 days. 
The rat must synthesize mitochondria with vary- 
ing levels of electron transport enzymes, rather 
than simply losing the enzymes from the mito- 
chondria. Perhaps the actual mechanism is a 
combination of both synthesis of incomplete mito- 
chondria and loss of enzymes from the mitochon- 
dria during development of the protein deficiency. 

Eefeeding the protein depleted rats with an 
adequate diet results in a rapid rate of liver cell 
synthesis. Within five days, the total number of 
cells have been restored but the cells are as low 
in nitrogen as they were prior to the repletion 
program. The formation of new protein-poor 
cells takes precedence over the repletion of the 
protein in the cells. This contrasts with the situ- 
ation in the rats pair-fed an adequate ration for 
the 72 days of the depletion part of the experiment. 
These animals also had a reduced number of liver 
cells but when fed on an ad libitum basis, they 
showed no increased production of liver cells until 
much later than was observed with the protein 
deficient rats. 

Maximum changes in many of the variables did 
not occur until 30 days after the start of the de- 
pletion regimen. The slow rate at which maximal 
changes occurred in both the protein-depleted rats 
and their pair-fed controls emphasizes the im- 
portance of maintaining animals and subjects on 
a dietary program for sufficiently long periods if 
it is desired to study the situation as it exists when 
equilibrium has been established. 

Experimental Obesity 

Genetic studies (with Dr. L. Sokoloff) with 
various strains of mice indicate that the gene gov- 
erning osteoarthritis was separate from those 



which controlled body weight and plasma lipids. 
Plasma total lipids, phospholipids and cholesterol 
levels showed little evidence for recessiveness or 
dominance of the genetic factors. 

A hybrid strain of rats (S5B/N) does not be- 
come obese when fed the high fat diet which made 
both parent strains obese. Lipid (FFA) mobili- 
zation by the hybrid rats was much less than that 
of the parent strains during fasting or following 
epinephrine injection. Blood glucose levels re- 
spond to these conditions in all three strains of 
rats. 

Marked increases occur in the concentration of 
total lipids, neutral fats, cholesterol and phospho- I 
lipids in the plasma of scorbutic guinea pigs. A 
decrease in the ester to total cholesterol ratio was 
found in scorbutic but not in fasting guinea pigs. 

Germfree Animal Research 

Germf ree rats and mice require a dietary source 
of folic acid whereas conventional animals receive 
adequate amounts of this vitamin from their bac- 
terial flora, except when fed diets very low in 
protein or when subjected to some stress such as 
administration of sulfa drugs. By inoculation of 
germfree rats with single strams of bacteria {E. 
coli, Aerohacter, Alkaligenes and Proteus) suffi- 
cient folic acid is supplied to the animal to prevent 
or cure folic acid deficiency. Wlien copi-ophagy 
is prevented in these monoinfected rats, there is 
still sufficient folic acid formed and made avail- 
able to the rat to cure a folic acid deficiency in- 
dicating that coprophagy is not necessarj^ to main- 
tain a flora or to obtain vitamins synthesized by 
this flora. Studies are in progress to determine 
what contribution, if any, are made by the oral 
flora with respect to vitamin synthesis. 

Experiments are in progress to study the devel- 
opment of dietary liver necrosis in the rat with 
both Tourula yeast diets and low protein diets, 
and to determine what efl'ects, if any, bacteria may 
have uj^on the relative activity of various forms 
of selenium with respect to prevention of necrosis. 

Experiments have been initiated with Dr. Pear- 
son of the University of California, Los Angeles, 
to study experimental arthritis in rats with respect 
to any role played by bacteria. 

The effect of biotin on the synthesis of folic acid 
by the germfree rat and the relationship of vita- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



189 



mill Bi2 and folic acid in the germfree rat is being 
studied. 



Guinea Pig Nutrition 

The guinea pig shows a normal rate of growth 
when either casein or a purified soybean protein 
are fed at a dietary level of 60%. At a level of 
70%, animals fed the soy protein ration showed 
excellent growth whereas tliose fed the casein ra- 
tion gained weight slowly and a number of them 
died. Tests are under way to determine whether 
the high methionine content of the casein pro- 
duces an amino acid imbalance. 

The methionine requirement of the growing 
guinea pig was shown to be 5.75 g/kg of diet. This 
is much lower than that required by most other 
animals. The guinea pig also differs from other 
animals in its reduced ability to utilize the D- 
isomer of methionine for growth. 



Glucose Tolerance Factor (GTF) 

Previous work showed that small amounts of 
insulin were required to elicit a "chromium re- 
sponse," the increase in glucose uptake by muscle 
in response to added trivalent chromium ions. It 
has recently been shown that glucose uptake and 
galactose entry rates into rat diaphragm and adi- 
pose tissue cells were not increased by low levels 
of insulin — such increases occurred only when 
small amounts of chromium were added to the 
insulin containing media. Although small 
amounts of chromium were detected in commercial 
insulin samples, replacement of the zinc with 
chromium produced a reduction in the activity of 
the insulin. Further evidence that chromium and 
insulin do not interact directly came from the find- 
ing that glucose uptake by epididymal fat pads 
was increased only when the two substances were 
added separately. 

Although chromium increases the amount of 
insulin bound by adipose tissue cells, it would have 
to do so four times more if this were its only 
action. It is suggested that chromium enhances 
the effect of the insulin, possibly as a glucose trans- 
port factor. 

A paradoxical phenomenon has been discovered 
in rats fed certain purified diets. The animals 
respond to an intravenous glucose tolerance by 



maintaining blood sugars of 200 to 300 mg% for 
hours. This probably results from the release of 
glucose by the liver. 

Studies are mider way on the assay and isola- 
tion of a water-soluble, heat-stable factor in liver 
which can function in the eviscerated, liverless, 
GFT deficient rat. 

High Calcium Intakes and Hemoglobin Determi- 
nations 

In a study of the efficacy of dietary phosphate 
supplment as a means of reducing the incidence 
of dental caries in the human, large amounts of 
calcium were also added to the diets of the children 
imder study. Since various reports of animal 
work indicated that high intakes of calcium inter- 
fered with the absorption of a number of trace 
elements, especially iron and copper, it became 
important to check such parameters as hemoglobin 
levels in the children. The cyanmethemoglobin 
procedure was chosen for this purpose since it was 
reportedly the best for field studies. The first 
results suggested that many children in both the 
supplemented and control groups had low hemo- 
globin levels. After extensive laboratory checks 
it became apparent that the cyanide-ferricyanide 
reagent became decolorized when frozen and re- 
mained so even after thawing. During the field 
studies, the reagent was usually kept in a station 
wagon where it could have frozen due to the low 
external temperatures. The reaction produced by 
freezing involved the reduction of the ferri- to 
ferrocyanide and the oxidation of the cyanide to 
cyanate. 

Studies carried out under conditions which elim- 
inated the above difficulty showed that even 
though the children received as much as 2.5 g of 
calcium per day for almost two years, their hemo- 
globin levels were the same as those of the chil- 
dren in the control schools. This suggests that 
high intakes of calcium added to a diet typical of 
that found among the poorer segments of the 
American population has no effect on the absorp- 
tion of those minerals involved in hemoglobin 
formation. 

Riboflavin 

Previous workers have reported a correlation 
between the urinary excretion of riboflavin and 



190 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



nitrogen during periods of negative nitrogen bal- 
ance. Both animals and man show pronomiced 
increases in urinary riboflavin excretion starting 
■with the first 24 hour urine samples collected dur- 
ing a starvation period. Preliminary work in this 
laboratory suggested that when human subjects 
were starved for periods of 18 hours, there was 
no increase in riboflavin excretion. For this rea- 
son, studies were initiated to determine when the 
increase in excretion occurs and its relation to 
nitrogen excretion. Fasting schedules ranging in 
duration from 29 to 45 hours have been followed 
by normal adults. Preliminary evidence reveals 
a 2- to 4-fold increase in riboflavin excretion about 
18 hours following the beginning of a fast, though 
with some individuals this increase is not seen until 
30 hours. These data will be correlated with 
nitrogen excretion. 

Adipose Tissue Metabolism 

A teclxnique has been developed for perfusing 
isolated adipose tissue from the parametrium of 
the rat. Wlien this tissue was removed from fed 
rats no free fatty acids (FFA) were released dur- 
ing a two-hour perfusion. Adipose tissue removed 
from fasted rats released FFA at a rate of 4.5 
microequivalents per gm during the first hour 
and 2.0 during the second hour. Addition of small 
amounts of insulin to the perfusate (2mU per ml) 
immediately arrested the release of FFA, even in 
the absence of glucose. Addition of glucose (240 
nig%) had no effect on the release of FFA. 
Epinephrine stimulated the release of FFA into 
the blood stream but to see this effect it was neces- 
sary to use a vasodilating drug such as papaverine 
(the latter had no effect on FFA release). Caf- 
feine enhanced and prolonged the effects of epi- 
nephrine. Caffeine also stimulated lipolysis. Adi- 
pose tissue taken from rats pancretectomized for 
one day released FFA twice as fast as adipose 
tissue from unoperated controls. 

Development of the teclmique for perfusing adi- 
pose tissue provides another method for studying 
the metabolism of body fat. This procedure not 
ony facilitates study of the isolated tissue but 
makes possible study of the role of the vascular 
bed in the response of the tissue to hormones, espe- 
cially to those that have pronounced effects on 
blood vessels, such as epinephrine and norepi- 
nephrine. 



Studies with incubated fat pads liave shown 
that metabolism of glucose by adipose tissue is } 
decreased in rats fed a diet which causes obesity. 
This diet, which has a high fat content, also de- 
creases the sensitivity of the fat pad to insulin as l| 
measured by uptake and conversion of radioglu- 
cose to CO2 and lipids. Efforts are being made to 
determine the reason for the difference in behavior 
of adipose tissue between rats fed the high fat 
and those fed a high carbohydrate diet. 

Glucocorticoids injected into hypophysectom- 
ized-pancreatectomized rats produced severe keto- 
sis, hyperlipemia and fattj' livers. Incubation 
studies with adipose tissue have confirmed the 
suggestion made earlier, as a result of the above 
findings, that glucocorticoids may have a direct 
action on adipose tissue. Addition of very small I 
amounts of glucocorticoids such as dexamethasone 
or triamcinolone to the incubation media (0.02 y 
per ml) increased the rate of FFA release and 
decreased the uptake of glucose. 



Protein Hormones 



4 



To date no one has succeeded in isolating a pure 
homogeneous thyroid stimulating hormone 
(TSH) preparation. The previous concentrates 
secured in this laboratory have contained several 
active components differing in their mobilities in 
starch-gel electrophoresis. Recent work confirms 
this and supports the earlier findings that TSH 
can exist in several active forms. A TSH prep- 
aration assaying 34 U per mg has been secured 
by purification on DEAE-C columns. 

The liormonal activity of TSH preparations is 
unaffected by prolonged papain digestion. Physi- 
cal measurements indicate that such treatment 
breaks very few peptide bonds. The activitj' is 
relatively stable in a solution at jiH 2.6 if the tem- 
perature is 2° C but at 25° C, 90% of the activity 
is lost within five hours. Considerable activity 
remains after standing at pH 11 for three hours 
at room temperature but spectropliotometric data 
suggest that some alteration of the protein 
occurred. 

Preparations of human TSH with an activity 
of 2 to 3 U per mg have been obtained by gel filtra- 
tion on Sephadex and chromatography on CMC 
carboxymethyl-cellulose. Tliis work indicated 
that human TSH may be more acidic than is the 
bovine preparation. The plasma of normal young 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



191 



men contains 1 to 12 milliunits of TSH per 100 
ml. Plasma from patients with simple goiters or 
thyroiditis had normal TSH levels while hypo- 
thyroid patients had levels of 40 to 100 milliunits 
per 100 ml. Hyperthyroid patients had normal 
or elevated levels (up to 40 milliunits per 100 ml 
plasma) . 

Rats with transplantable pituitary tumors 
(MtT) have in their blood a 100 times higher 
concentration of growth hormone and prolactin 
than normals and 10 times more ACTH. Thyro- 
tropin and gonadotropin levels are apparently not 
elevated. Animals with these tumors show a 10 
to 20% increase in body weight, a 50% increase 
in gut weight, a 3-fold enlargement of liver and 
kidney, a doubling of heart weight, a 5-fold in- 
crease in preputial gland weight and mammary 
glands fully developed and filled with milk. On 
the other hand, the thymus and body fat stores 
are absent and the ovaries and uteri are half 
normal size. The preceding changes are sunilar 
to those seen in hypophj^ectomized pigeons in- 
jected with growth hormone, prolactin and ACTH. 
Similar changes are not seen m the rat under 
comparable conditions probably due to the fact 
that sufficiently high blood levels are not attainable 
in this species by injection. 

There was 50 to 100 times as much growth hor- 
mone and ACTH in the tumors as in the blood 
while there was only twice as much prolactin in the 
tumors. This finding is compatible with the 
theory and data of others that the hypothalmus 
inhibits release of prolactin and contact with the 
hypothalmus is necessary to permit storage of pro- 
lactin in pituitary tissue. 

The adrenals of rats with the transplanted MtT 
tumor increase in size about the time the tumor 
begins to appear. At this time, the adrenals no 
longer respond to stress or ACTH injections with 
elevated blood levels of corticosterone and deple- 
tion of adrenal ascoi'bic acid. The high blood level 
of endogenous ACTH has the adrenal functioning 
already at masimmn capacity. 

Incubation of the adrenals of rat fetuses in a 
Krebs-Ringer solution produced corticosterone in 
proportion to the amomit of ACTH added. The 
corticosterone production per mg of adrenal was 
much higher for the fetal adrenal than for the 
adrenals from 250 g rats suggesting a five times 
greater sensitivity in the former to ACTH. 



LABORATORY OF PHARMACOLOGY AND 
TOXICOLOGY 

Spermidine and Spermine 

These polyamines have been investigated in this 
laboratory for several years in collaboration with 
Dr. S. M. Rosenthal. They are widely distributed 
in a variety of biological materials, and previous 
reports have been concerned with their distrib- 
ution, biosynthesis, and metabolism. 

Recently, our work has been concerned with the 
stabilizing effects of these amines on a variety of 
biological materials. These polyamines will stabi- 
lize protoplasts, spheroplasts, bacteriophage, and 
deoxyribonucleic acids. These stabilizing effects 
have been shown against a variety of agents, in- 
cluding hypotonic lysis, heating and mechanical 
stress (shearing). Very low concentrations of the 
polyamines are effective, and suggest that these 
stabilizing effects are indicative of a physiologic 
function for these amines. The most likely mech- 
anism for these effects is the formation of a com- 
plex between the amine and both phospholipids 
and nucleic acids. 

We have also continued our studies on the bio- 
synthesis and metabolism of the polyamines. As 
part of these studies, a new enzymatic reaction has 
been described in extracts of Serratia marcescens 
that converts spermidine stoichiometrically to A'- 
pyrroline, 1, 3-diaminopropane, and H2O2. 

Sialic Acids 

The term, sialic acid, refers to a group of com- 
pounds tliat are derivatives of neuraminic acid, 
a 9-carbon carbohydrate. They are usually found 
linked to various carbohydrate polymers, and, as 
indicated in part by the work smnmarized here, 
appear to be of importance in a variety of biologic 
areas. 

. One example of the sialic acids is i\^-acetylneura- 
minic acid, and a major contribution during the 
past year has been the elucidation of the enzymatic 
biosynthesis of 7V-acetylneuraminic acid. Three 
separate enzymes have been purified from rat liver 
and bovine submaxiallary gland, which carry out 
the following reactions : 

1. N - Acetyl - D - mannosamine+ A T Y-^N- 

acetyl - D - mannosamine- 6-phosphate-l- 

ADP 



192 



ANNUAL REVIEW OF INTEAMURAL RESEARCH 



4 



2. iV^- Acetyl - D -mannosamine-6-pliosphate+ 

phosplioenolpyruvate -» N - acetylneura- 
minic acid-9-phosphate+Pi 

3. iV-Acetylneuraminic acid-9-pliosphate->7V- 

acetylneuraminic + Pi 

Another aspect of these studies has been con- 
cerned with sialidase, an enzyme that splits the 
sialic acid moiety from the polymer to which it is 
attached. The enzyme has been purified from 
plasma proteins. Sialidase activity has also been 
found in purified diphtheria toxin, and is inhibited 
by diphtheria antitoxin. Sialidase has also been 
detected in rat thyroid (with Dr. S. WoUman) . 

The sialic acid studies have also included (1) 
histochemical studies of sialomucins in human 
mammary carcinoma with Dr. S. S. Spicer, (2) a 
phylogenetic study of sialic acid distribution, (3) 
discvovery of new forms of sialic acid in star fish 
and sea urchin eggs. 

Sulfur Amino Acids 

Previous work from this laboratory has pre- 
sented evidence that three enzymatic steps are re- 
quired for the reduction of methionine sulfoxide 
by TPNH. It has now been shown that at least 
one of these enzymes (and possibly two) are 
flavoproteins. 

During this study it was also found that yeast 
glutathione reductase is also a flavoprotein con- 
taining — SH groups; this aspect of this well- 
known enzyme had not been previously observed. 

Enzyme Levels 

Considerable work has been reported from vari- 
ous laboratories on the factors controlling the level 
of various enzymes in bacterial systems. Very 
little work, on the other hand, has been done on 
these control mechanisms in mammalian systems. 

We have now been able to demonstrate clearly 
that each of the enzymes involved in urea synthe- 
sis (carbamylphosphate synthetase, ornithine 
transcarbamylase, arginosuccinate synthetase and 
cleavage enzyme, and arginase) increases when 
the protein intake and urea excretion increase, 
and decreases when the protein intake decreases. 
Various studies have been carried out, including 
enzyme isolation, to show that these changes re- 



flect actual changes in enzyme levels, and are not 
the result of activators or inhibitoi-s. 

Isotopic studies have shown that the enzyme 
level is regulated by active control of both enzyme • 
and synthesis and of enzyme degradation. 



Burns 

Both laboratory and clinical studies on bums 
have concentrated mainly on the role of infection, 
since the work of the past few yeai-s has indicated 
how important infection is in the mortality follow- 
ing burns. In the Peru project PseudomoTMS has 
been the organism most often involved. 

In the laboratory a potent antiserum against 
Pseudomonas has been developed, and has been 
shown to be effective in vivo against 23 different 
isolates of Pseudomonas aeruginosa. In other 
studies preliminary experiments have shown (1) 
an increase in survival of burned mice upon treat- 
ment with convalescent serum and (2) the presence 
of an antigen in burned mouse serum different 
from those f omid in normal serum. 

Associated with these laboratory studies, similar 
data are being collected from clinical burns in the 
Peru project. Since Pseudomonas infections were 
seen frequently in these cases, the cases were 
treated prophylactically with gamma globulin, 
plasma, or albumin, and tlie effect of these treat- 
ments noted on early and late mortality. No 
effect above that due to saline solution alone was 
noted in adults, but in burned children a definite 
protective effect was observed with gamma globu- 
lin and plasma that was not observed with albu- 
min. Pseudomonas antiserum is also being tested 
in the therapy of human burns, but insufficient 
data are available yet on the efficacy of this anti- 
serum. At present, the preferred treatment for 
burns in young children is saline solution plus 
plasma and gamma globulin. 

Histidine, Histamine, and Related Imidazoles 

The most interesting development in this area 
has been the demonstration by exchange reactions, 
that an amino-enzyme is an intermediate in the 
reaction: Z-Histidine->urocanic acid -f- NH; 
In addition, further purification studies have been 
carried out on this enzyme, as well as on urocanase 
and diamine oxidase. 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



193 



Further work has also been carried out on the 
identification of the imidazoline and imidazolidine 
compounds that have been obtained by the cata- 
lytic reduction of imidazole compounds in acetic 
anliydride. These are new derivatives as this re- 
duction represents a new chemical reaction. The 
organic chemical studies on the synthesis and 
characterization of the ribosides of imidazole- 
acetic acid and other imidazole compoimds has 
also been continued. 

Leprosy 

The chemotherapeutic studies on mouse leprosy 
that have been conducted for many years are con- 
tinuing, but have now been supplemented with 
chemotherapeutic studies on human leprosy. For 
the latter studies, in collaboration with Dr. C. 
Shepard, C.D.C., the technique growth of or- 
ganisms in the mouse footpads has been used. 

In the mouse studies a new phenazine derivative 
has been the most active chemotherapeutic drug 
yet f oimd. Two ethylmercaptan derivatives were 
also studied ; these drugs were almost as effective 
as isoniazid, but showed the additional advantage 
in slower development of resistance. 

The human leprosy studies show that diamino- 
diphenylsulfone, isoniazid, para-aminosalicylic 
acid, and cycloserine were effective; studies of 
other agents are in progress. 

Improvements have also been made in the con- 
ditions for growth of Mycobacterium leprae mu- 
rium, the agent of mouse leprosy. 

Mevalonic Acid 

Studies in this area have been directed towards 
discovering new pathways for mevalonic acid utili- 
zation. As part of these studies Lactobacillus 
acidophilus has been grown on C^* -mevalonic acid, 
and a large incorporation of label has been dem- 
onstrated in an imidentified neutral lipid, contain- 
ing a free hydroxyl group, that does not appear to 
be one of the common steroids. 

Enzyme Activity 

Hydroxylysine has been previously reported by 
Drs. Viswanatha and Irrevere as a constituent of 
trypsin and chymotrypsin. An activating enzyme 



(detected by pyrophosphate exchange) for hy- 
droxylysine has now been found in pancreatic and 
yeast extracts. 

Enzymatic Conversion of Vitamin B12 to Its 
Coenzyme Form 

Vitamin B12 has been enzymatically converted 
to its active coenzyme form by an enzyme from 
Clostridium, tetanomorphuon in a reaction mixture 
containing glutathione, ATP, and yeast extract 
(in collaboration with Dr. H. Weissbach of the 
National Heart Institute) . 



LABORATORY OF PHYSICAL BIOLOGY 

Basic Cellular Induction Mechanisms 

Dr. Park's discovery that sexual differentiation 
in Hydra may occur cyclically under uniform 
mass culture conditions and that sexuality is not 
specifically induced by COo, bicarbonate or ver- 
sene buffer, or by aeration was shown to be true 
in a second strain of Hydro. Stirring the culture 
8 hours per day has been shown to prevent dif- 
ferentiation for at least 4 weeks. 



Protein Synthesis in Insect Metamorphosis 

Citrate oxidation is shown to be indicative of 
Krebs cycle activity in adult development. It is 
about three tunes as great after emergence as just 
prior to emergence. During adult development 
there is a nine-fold change in citrate oxidation 
compared with only a three- fold change in citrate 
turnover. A study of blowfly muscle amino acid 
composition shows tropomyosin from larval and 
adult flies to be similar but to have different phys- 
ical properties. 

Factors Influencing Insect Respiration 

The alkaloid ryanodine has been shown to: 
stimulate respiration in roaches and grasshoppers 
but not in adult flies ; paralyze fly leg muscles but 
not wing muscles ; and to cause flaccid paralysis in 
the legs of flies but tonic paralysis in fly larvae. 
Cole exposure which easily damages the mecha- 



194 



ANNUAL REVIEW OF ESTTRAMURAL RESEARCH 



nisin responsible for pujDation of bee-moths is 
shown to be ineifective in army-work larvae. 

Biological Triggers 

An extensive review of the biophysics of insect 
respiration and the first paper of a series on trig- 
gering of firefly flash have been completed. 

Physical Factors Influencing Breathing 

Pressure-flow lag shown in earlier work to exist 
in active breathing patterns was found to be in- 
fluenced by subjective "effort" in carrying out tlie 
breath cycle. A mechanism is not yet known for 
this phenomenon. 

Physiological States That Affect Tolerance of 
Hypoxia 

It is seen that prolonged exercise and subjection 
to hypoxia each bring about clianges in serum 
enzyme levels which may be related to pathologic 
changes. Such relationships may aid in the diag- 
nosis of diseases through exercise tolerance stud- 
ies where biopsy may be impossible or unde- 
sirable. 

Circulatory Reaction to Plasma Expanders 

Edema does not form in anesthetized rats after 
minimal dextran doses in the presence of hyper- 
glycemia although the blood pressure is normal. 
Significant edema occurs in conscious rats with 
vaso-depression but no hyperglycemia. Primary 
factor in transudation of fluid thus is blood glu- 
cose level rather than hemodynamic condition. 

Evaluation of Molecular Organization at Inter- 
faces 

In monomolecular films of lipids containing 
phosphate groups, but not those containing — OH, 
— NH, or ester groups, procaine acts as a crosslink- 
ing agent with the ionic portions of the film lipids ; 
veratrine on the other hand reacts with the lipid. 

Muscle Fiber Energetics 

Eefined methods of measuring the ATPase 
activity of glycerol-treated muscle fibers have re- 
vealed that the activity is almost linear with time. 
The diffusion of ATP into fiber-bundles was found 



to be about 0.5 x 10"** cm-/sec., 100 times gxeater 
than the formerly accepted value. 



Muscle Proteins 



4 



Evidence has been presented showing that the I 
tropomyosin-like component of mj'osin is the an- 
alogue of the paramyosin of invertebrate muscle. 



4 



Enzyme Reconstitution 

Studies on pyruvate and alpha-ketoglutarate 
dehydrogenase complexes from Escherichia coU 
show that subunits separated in various ways can I 
be recombined to restore the original "multi-en- 
zyme" units with the natural activity. 

I 

Small Ion Binding in Tertiary Structure of * 

Proteins 

From ion binding studies it has been shown that 
the peptide chain of ribonuclease is folded in a 
manner to create two clusters containing two im- 
idazol groups and one ammonium (or giianidini- 
um) group in each cluster, each cluster thus acting 
to hold the claain in a specific fold. 

Genetic Differences in Bovine Beta-lactoglobu- 
lins A and B 

By amino acid analysis and "peptide mapping" 
it was shown that bovine ^-lactoglobulin A. differed 
from /?-lactoglobulin B in two residues. Since no 
lactoglobulin differing in a single amino acid has 
been discovered, it is suggested that this double 
substitution may have arisen from a single muta- 
tional event. The possibilitj' of two successive 
mutations with loss through selection of the inter- 
mediate form can not be I'uled out. 



Blood Proteins 



A detailed study of both bovine and human 
thrombin by ultracentrifugal assays have shown 
a very low sedimentation rate for the DFP-in- 
activated thrombin confirming the low molecular 
Aveight previously reported from this laboratory. 
The entire sequence of peptide B of Co-fibrin lias 
been elucidated. It contains 21 amino acids in a 
single polypeptide chain, in which the free alpha- 
amino group is blocked by N-acetyl substitution. 
It was found possible to oxidize the carbohydrate 
component of fibrinogen by periodate witliout 
destroying an_y of tlie amino acid residues and such 



^ 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



195 



treatment renders fibrinogen incapable of poly- 
merization, indicating that the carbohydrate 
moiety is essential in keeping the fibrinogen clot- 
table. 

Gas Chromatography of Amino Acids 

Chromatograpliic procedures for the analysis of 
the amino acids have been under continued study. 
Ammonia has been introduced as a carrier gas 
to make practical the use of the ester in gas phase 
analysis. 

Unusual Amino Acids Isolated From Biological 
Materials 

The quest for novel amino acids in natiu'al prod- 
ucts has been continued. S-hydroxyproline in 
sponge and in the antibiotic Telomycin was 
demonstrated. 

Macromolecular Protein Synthesis in Yeast 

The use of fluorouracil, an analog of the nox-mal 
metabolite uracil, demonstrates that the so-called 
"inner pool" of amino acids which are precursors 
of the proteins built by the yeast, differentially 
accumulates four of the acids without effect on the 
protein synthesis other than rate or total quantity 
produced. This fivefold accumulation is of inter- 
est in delineating the possible template mechanism 
permitting such behavior. 

Cellular Reaction to Sera From Carious Disease 
States 

Hela cells adapted to pooled human sera are 
foimd to madergo recognizable form changes in the 
presence of test sera from a variety of patients 
whose diagnosed disease state falls into the cata- 
gory of renal diseases and autoimmune conditions. 
Investigation of the possible factor or factors in- 
terfering with the normal growth of Hela cells is 
mider way. 

Transmission of Light by the Crystalline Lens 

Both transmitted and scattered light studies of 
the rabbit lens have been made showing that back- 
scattering increases with age and that diffuse opac- 
ity developed m tissue culture of such lens is due 
to scattermg losses from the transmitted beam. 
The physics of the scattering process pertinent to 
the crystalline lens has been reviewed indicating 



that a paracrystalline state of the soluble lens pro- 
teins can be inferred to explain transparency and 
that the larger proteins of the cell walls minimize 
scattering by their regular spacing. 

Source for Neutron Irradiations 

Irradiation in a mixed flux of known composi- 
tion has been made available. Its use for activa- 
tion analysis either with fast neutrons or thermal 
neutrons is feasible. Dosimetry for use in irradia- 
tion studies have been worked out. 

Graded Electron Beam Ionization Effects on 
Protein Denaturization 

Basic globular proteins subjected to dry, in 
vacuo, electron bombardment acquire the ability 
to induce excess turbidity in polynucleotide solu- 
tions. This turbidity is characterized as a non- 
monotonic, protein characterizing function of the 
ionizing radiation dose. A comparison of the 
functions obtained for chymotrypsinogen, ribo- 
nuclease, and lysozyme shows that the isoelectric 
pH of, the molecular weight of, and the number 
of sulfur bridges possessed by a protein are im- 
portant determinants of the differences observed. 

Electrochemistry of Membranes and Interfaces 

Thermosmosis, the transport of liquids across 
a membrane wliich separates two liquid phases of 
identical composition but unequal temperature has 
been re-examined. The process requires the pres- 
ence of electrolytes and electrically charged mem- 
branes. It is thus shown to be a special case of 
electro-osmosis. It has been shown also to be strik- 
ingly similar to the same parameters which con- 
trol anomalous osmosis. A study of silicon car- 
bide crystal electrodes which should be highly 
inert and independent of the composition of the 
solutions to be measured has been made to test the 
feasibility of their use as reference electrodes. The 
loss of photovoltaic sensitivity and its restoration 
by anodization seem to control the required prop- 
ei-ties of these crystals as reference electrodes. 
Studies of collodion matrix ion-exchange mem- 
branes were extended to test the "contractility" 
effect. A model was considered which satisfac- 
torily accounted for the variation of electro-os- 
motic permeability with concentration; it passes 
through a maximum as the polyelectrolyte uncoils 
or swells in the membrane pore. Studies of the 



196 



ANNUAL REVIEW OF INTEAMURAL RESEARCH 



nonstructural homogeneous (oil) membranes have 
progressed despite great experimental difficulties 
to show both cation and anion selective oil mem- 
branes. The low conductivity of the oil mem- 
branes indicate that the ions which permeate 
across the membrane must be present in the oil in 
a non-ionized form. 

Macromolecular Organization of Substances of 
Biological Interest 

The analysis of the crystal structure of protein 
crystals using electron micrographs together with 
models has been applied to the diamond shaped 
parasporal bodies formed in Baccillus Thurin- 
giensis showing them to be essentially composed 
of cubic close-packed spheres about 80 A in diam- 
eter which form a face centered cubic structure 
having a tetramolecular unit cell of about 110 A 
on an edge. Analysis of the periodicities appear- 
ing on organic crystal images in the electron mi- 
croscope show them to be associated with a single 
resolvable molecular plane, but the condition at- 
tending the microscopy may show dimensions 
which may be any of several multiples or fractions 
of the true value. An analysis of these effects has 
been made for the dye indanthrene olive T. 

NMR Characterization of Molecular Structure 

Nuclear magnetic resonance has now estab- 
lished itself as a powerful spectroscopic technique. 
During the past year this was used to extract in- 
formation of fundamental importance to the eluci- 
dation of the molecular structure of a series of 
porphyrins and lignans and permitted the exclu- 
sion of certain alternative interpretations. Simi- 
larly the technique was an invaluable aid to the 
structural elucidation of a number of naturally 
occurring alkaloids, steroids, and amino acids. 

Optical Activity of Molecular Structures 

The origin of the major contribution to the 
optical activity of the optical isomers of organic 
compounds containing a conjugated diene system 
has been shown to be due to the diene itself if the 
four carbon atoms of the diene are skewed out of a 
single plane. On the basis of a ntraiber of such 
substances studied it has been shown that several 



types of compounds not before amenable to in- 
vestigation by the method of rotatoiy dispersion 
can now be studied. The possibility of elucidat- 
ing absolute configurations of dienic compounds 
by a simple measurement is of particular 
significance. 

Ergosterol-Vitamin D Mechanism and Other 
Photochemical Studies 

Data representing the only comprehensive in- 
vestigation of the action of monochromatic radia- 
tion on ergosterol and its photoisomers are being 
analyzed. Experimentation with the recording 
of total information on magnetic tape has been 
initiated and will be extended to other sources of 
data such as gas chromatography and ultraviolet 
absorption. Considerable interest attaches to the 
limitations on computor apj)roach to these deter- 
minations. The effect of structural changes in 
substituted diazophenones on photochemical con- 
stants has been investigated, and consideration of 
the molar extinction coefficient yield an equation 
which permits the development of a theoretical 
and mechanistic basis for this relationship. 

Structure as Related to Chemosynthesis by 
Radiant Energy 

The use of image converters for direct observa- 
tion in the infrared spectral region and polarizing 
optics to detect polarized fluorescence and dichro- 
ism have yielded direct evidence of a high degi-ee 
of orientation of the pigment molecules in the 
chloroplast of Euglena. The significance of the 
laminar structure in this and other biological 
structures is seen to be critical to the specific 
biological fimctions dealing with energy transfer. 

Bioelectric Phenomena of Photoreceptors 

Microelectrode recordings from isolated slices 
of squid x-etina during direct observation via 
image converters in infrared light have permitted 
the examination of dark and light adapted imits 
to demonstrate that the mechanism of excitation 
is a depolarization of the whole photoreceptor by 
a flow of membrane current through only that 
part of the cell which is illuminated. Effects of 
light adaption and the relationship of sodium and 



11 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



197 



potassium ions to the membrane currents have also 
been studied. 

Temperature Dependence of Form and Viru- 
lence of Hemo flagellates 

Studies on a number of parasitic hemoflagel- 
lates has shown that where there is a different 
optimum temperature for the development of the 
form the host tissues attacked will be those nor- 
mally carrying the optimal temperature without 
regard to the type of host cell. Temperature is 
also seen to affect the organization of the outer 
form of the organism and it is inferred that simi- 
lar effects on internal organization account for the 
activity and viability of the cell. 



OFFICE OF MATHEMATICAL RESEARCH 

Work lias contiuued along the broad lines indi- 
cated in the report of last year : Development of 
mathematical and computational methodology for 
mathematical models based on differential equa- 
tions; mathematical formulation and analysis of 
neurophysiological problems ; general mathemati- 
cal problems arising from the rate behavior of 
biological systems ; mathematical studies of visual 
perception. Progress in these areas is summarized 
below in terms of highlights and examples. 

Solutions have been obtained for a set of prob- 
lems designed to answer the question : Is the con- 
tribution made to the integrative properties of a 
neuron by synapses distributed over the dendritic 
surface significant and, if so, what are the effects 
of various dendritic distributions of synaptic 
excitation and inhibition ? The solution of a mul- 
ti-region boundary-value problems, based upon a 
transformation reducing the appropriate differen- 
tial equations to a canonical form and expression 
of synaptic activity as step conductance changes in 
the nerve membrane, yields the transient potential 
distribution for any assumed distribution and rela- 
tive intensities of excitation and inhibition. Quan- 
titative calculations suggest that dendritic synap- 
tic activity dominates the slowly changing back- 
ground level of neuronal excitatory state, while 
central synaptic activity produces rapid soma 
changes especially appropriate for "triggering" 
of impulse discharge by the neuron. Independent 



experimental support for this suggested functional 
distinction has recently been obtained by the 
neuro-anatomy group at Oslo. The method of 
computing extraneuronal action potentials, re- 
ported last year, has been extended to include the 
time course as well as spatial dependence and pro- 
granmied for the IBM-650 digital computer. Cal- 
culations, based on assumed passive dendritic 
membrane, give predictions m excellent agreement 
with the observations of Drs. K. Frank and P. 
Nelson (NINDB). Previously the shape of the 
transient action potential curves and apparent 
conduction velocities have been claimed to sup- 
port the assertion that nerve impulses are propa- 
gated into the dendritic tree. The remarkable 
agreement of experiment with predictions based 
on a theory explicitly denying this assertion calls 
for a reconsideration of this question and its not 
inconsiderable physiological implications. (Dr. 
Wilfrid Rail) 

The computer program for routine analysis of 
data and the formulation and assessment of models 
has been extended to handle larger (more varia- 
bles) and more complex systems. The method- 
ology has been improved to secure more rapid con- 
vergence for iterative solutions. A general 
formalism has been advanced for analysis of data 
in terms of linear compartmental systems which 
embodies much of the flexibility and versatility of 
analogue computer methods but which retains the 
precision, ability to assess uniqueness, goodness of 
fit, etc., of the digital computer program. A 
postulate regarding perturbations in linear sys- 
tems has been advanced and explored quantita- 
tively in hypothetical as well as actual systems. 
When a perturbation can be described by maximal 
changes in a minimal number of parameters, com- 
parisons of the original and perturbed system 
permit a unique prescription of the model when 
information on the original system alone is in- 
sufficient to do this. The above program and 
methodology have been applied to a number of 
problems in collaboration with other investigators. 
(Dr. Mones Berman and Mrs. Marjory Weiss) 

A formal model for brightness perception has 
been formulated and shown to give good agree- 
ment with experimental results under a wide va- 
riety of conditions. The essential features of the 
model are an averager, which measures average 
illumination, lo; a differencer which forms the 



198 



AJSTNTTAL REVIEW OF INTEAMURAL RESEARCH 



difference between lo and the illumination at any 
point; and a variable gain amplifier with gain 
linear in lo. Under certain circmnstances, in- 
creased illumination may increase, decrease or 
leave unchanged the perceived brightness of an 
object. This much discussed phenomena is ac- 
counted for qualitatively, in a simple way, by the 
brightness equations derived for the model which 
shows good quantitative agreement with experi- 
mental results from two independent sets of 
studies. The basic model can be refined by includ- 
ing a local averager, which yields well known edge 
effects, and a coupling capacitor, which yields the 
stabilized retinal effect. (Mrs. K. B. Marimont, 
NIMH, Associate member OME) 

It has been shown that if x(t) is a solution of a 
linear second-order differential equation with 
negative characteristic roots and exhibits a maxi- 
mum or minimum at t=T, then x(t) has the prop- 
erty that T |x(T)|/|A|^l/e where A is the area 
under the entire x(t) -curve. The inequality is 
strict unless x(0) =0 and the characteristic equa- 
tion has a double root. The theorem provides a 
means of distinguishing between critical and over- 
damping in any damped second-order system as 
well as a test of the hypothesis that a given chemi- 
cal or metabolic product is separated from the 
original precursor by two or more intermediates. 
It has also been shown that if a matrix, A, of order 
n 4-1 is sign-symmetric and of the form 



where D is diagonal of order n, then the roots of 
A are real and separated by the diagonal elements 
of D. Practical and theoretical consequences of 
this theorem in terms of blood-gas exchange, 
tracer kinetics and chemical kinetics have been 
derived including conditions for a repeated root 
and conditions under which a n*"" order system 
will behave as a system of order n-k, l^k^n-1. 
A theorem with closely related consequences is 
this: If u= (1, 1, . . ., 1) is an eigenvector of the 
matrix A then the sums along any row of the 
cofactors of adj(A — xl) have a common value 
for all X. This theorem establishes necessary and 
sufficient conditions imder which the smn of the 
species in a linear chemical or metabolic system 
will behave as a single species and under which the 



amount of an injected material remaining in the 
body at any time is independent of the site of 
injection. (Dr. John Z. Hearon) 



CLINICAL INVESTIGATIONS 

This is the first year that the number of beds 
(70) allocated to NIAMD and clinical units has 
remained constant. The patient census (occu- 
pancy) for 1961 has averaged 83%. 

A total of 480 in-patients was admitted during 
the 12-month period from December 1, 1960 to 
November 30, 1961. The total patient-days was 
19,856, an increase of 722 over the preceding year. 
In the Admissions and Followup Department 
1,881 patients were examined and studied, an in- 
crease of 296 over the past year. The average in- 
patient stay at the Clinical Center was 41 days. 

Investigations related to the diseases studied at 
NIAMD have resulted in 93 publications in scien- 
tific journals, monographs, annual reviews and 
medical textbooks. Dr. James Field and Dr. Ira 
Pastan were awarded the Van Meter Prize by the I 
American Goiter Association in recognition of 
their elucidation of the mechanism of action of the 
thyroid-stimulating hormone secreted by the an- 
terior pituitary. Dr. Paul di Sant'Agnese and his 
associates, Dr. Gibson and Dr. Landauer, were 
awarded a prize by the American Medical Associa- 
tion for their exhibit on Cystic Fibrosis of the 
Pancreas. Dr. Joseph J. Bunim gave the annual 
A.O.A. lecture at Yale University College of Med- 
icine. At the International Congress on Rheu- 
matic Diseases in Rome he was elected as Honor- 
ary Member of the Italian Rheumatology Society. 

Our staff scientists haA^e derived substantial 
benefit from the association of distinguished Visit- 
ing Scientists and Guest Workers who have 
worked at our Institute during the past year : Dr. 
John Lawrence of Manchester University, Direc- 
tor of Field Unit of Empire Rheimiatism Council ; 
Dr. Mario Andreoli of University of Rome, Italy ; 
Dr. Mehroo Bharucha of Basel University, 
Switzerland ; Dr. Tetsuo Shiba of Osaka Univer- 
sity, Japan ; Dr. Othmar Gabriel and Dr. T^wis 
Gibson, Fellows of the National Cystic Fibrosis 
Research Foundation; Dr. Harry Keen, PHS 
Fellow from England; Dr. IVIario Werner, Basel 
City Hospital, Fellow of Swiss Academy of Medi- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



199 



cal Science; Dr. Nicholas Halmi of Iowa State 
University, Fellow of National Science Founda- 
tion ; and Dr. David Matthews of Royal Free Hos- 
pital and School of Medicine, London, Fellow of 
British Postgraduate Medical Federation. 



ARTHRITIS AIND RHEUMATISM BRANCH 
Demography of Rheumatic Diseases 

The earliest studies on prevalence of rheumatic 
diseases were done only within the past 25 years. 
Most of these were sociologically oriented and are 
of little value to the biomedical investigator in- 
terested in gaining some insight into the causes 
and predisposing factors of rheumatoid arthritis, 
osteoarthrosis, systemic lupus erythematosus, gout 
and other rheumatic diseases. The principal de- 
ficiency in these prevalence studies is that they are 
based on subjective complaints of joint symptoms. 
Only since 1956 have reports appeared that were 
based on acceptable methods for determining 
prevalence in population surveys. In the recent 
surveys, prevalence of specified types of arthritis 
have been determined by objective and interna- 
tionally standardized criteria which include clin- 
ical findings, radiographic abnormalities, and 
serological tests for rhemnatoid factors. Seven 
such surveys have been reported to date in North- 
ern Europe (England, Wales, Scotland, Holland, 
and Finland) , and only one in the United States 
(Pittsburgh). These reports represent a promis- 
ing start, but as is to be expected from any pio- 
neering effort in clinical investigation, the results 
so far have served to sharpen the focus on certain 
specific questions which require that new studies 
be undertaken that are so designed and directed 
as to yield, hopefully, the desired information. 

The studies in which we are currently engaged 
have been designed to answer several questions. 
Based on standardized criteria and comprehensive 
examinations of a random sample of this nation's 
population, what is the prevalence of rheumatoid 
arthritis and osteoarthrosis in the U.S.? How 
does prevalence of these diseases vary with age, 
sex, geographic location, environment (urban and 
rural) ? What is the influence of age and environ- 
ment on the occurrence of rheumatoid factors in 



the serum of rheumatoid and non-rhemnatoid 
respondents? In population groups of similar 
racial, etlxnic, cultural and economic backgroimd 
(the American Indians) what effects do a widely 
varying climate (e.g. 54°, 48°, 33° and 15° latitude 
north) and altitude have on occurrence of rheu- 
matoid arthritis, osteorarthrosis, on erosive artic- 
ular changes (by x-ray) in cervical spine, 
sacroiliac joints, hands and feet, and on rheuma- 
toid factors ? Studies done in our laboratories and 
by others suggest that there are at least two rheu- 
matoid factors: one that complexes with human 
aggregated gamma globulin (demonstrated by the 
bentonite flocculation test), and another with 
antigen-antibody complexes (demonstrated by the 
sheep cell agglutination test) . There is evidence 
to suggest that the latter is genetically determined 
and the former is influenced by environmental 
factors. This hypothesis can now be tested by a 
family study on the Indian population now under 
survey by our unit. If the occurrence of abnormal 
circulating antibodies to tissue components — 
which some workers regard as evidence for de- 
velopment of auto-inmiunity — is a result of 
genetically deranged immunological mechanism, 
appropriate study of the relatives of patients with 
a rheumatic disease such as Sjogren's syndrome 
(which is associated with this phenomenon) should 
contribute significant information. This study has 
been undertaken by our unit and is near comple- 
tion (vide infra). 

^Vliat has been said thus far will serve as intro- 
duction to summaries of three studies now under- 
way in the Arthritis and Rheumatism Branch: 
(1) National Health Examination Survey for 
prevalence of arthritis, (2) survey of American 
Indians at widely different latitudes for preva- 
lence of arthritis, and (3) family studies on 
Sjogren's syndrome. 

NatioTial Examination Arthritis Survey 

The National Health Survey Division (Mr. 
Theodore Woolsey and Dr. Alice Waterhouse and 
staff) is currently conducting a Health Examina- 
tion Survey in which 42 representative areas se- 
lected in a stratified random sampling pattern 
throughout the U.S. will be visited by two mobile 
examining centers, each staffed by teams of 10 



200 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



persons and operating simultaneously in two spe- 
cially constructed, large truck trailers. A prob- 
ability sample of 6,000 adults aged 18 to 79 years 
selected from the population of 93 million civilian 
adults (non-institutional) will be examined. The 
survey was begun July 1960 and is scheduled for 
completion by July 1963. NIAMD is collaborat- 
ing in the arthritis component of this survey. 
The mobile team takes a complete history of mus- 
culo-skeletal complaints covering especially symp- 
toms of arthritis and rheumatism, and performs 
a standardized examination of the joints. Radio- 
graphs of both hands and feet are made and a 
blood sample taken. The x-rays and serum sam- 
ples are shipped to NIAMD in Bethesda. Ben- 
tonite flocculation tests are done in our laboratory 
and the films are read independently by three ob- 
servers (Drs. Bunim, Burch and Black) . To date 
(November 1961) 3,421 sermn samples collected 
from 26 of the 42 areas have been tested. Of these, 
118 (3.4%) gave a positive bentonite flocculation 
test. When the survey is completed more detailed 
analysis will be made and the relation of various 
environmental factors, as well as age and sex, to 
the presence of rheumatoid factor will be deter- 
mined. In the aforementioned surveys in northern 
Europe, the prevalence of positive tests for rheu- 
matoid factor (sheep cell agglutination test) 
varied from 2 to 5%, being higher in the urban 
areas than in the rural (P= <.01) . 

Thus far (in three areas completed) radio- 
graphic evidence of osteoarthrosis (grade 2 or 
higher) has varied from 28.1% in Valdosta, Geor- 
gia to 48.6% in Akron, Ohio. The frequency of 
changes consistent with rheumatoid arthritis 
varied from zero in Valdosta to 1.5% in Philadel- 
phia. (Drs. Bunim, Burch & Black) 

Indian Survey 

This survey was undertaken for several reasons. 
The principal one, to test the hypothesis that 
prevalence of rheumatoid arthritis and possibly of 
osteoarthrosis was related to climate. This rela- 
tionship first became apparent from results of 
recent population surveys in England and Wales 
(Lawrence 1960 and Miall 1958). Prevalence 
rates of rheumatoid arthritis in four towns going 
from north to south are listed below : 



Town 


Latitude 
(degrees) 


Prevalence Rheu- 
matoid Artlirltls 
(age 15 and over), 
percent 


Wensleydale, Yorkshire 

Leigh, Lancashire 


54-55 

54 

51-52 

51-52 


5.7 
3.9 


Glamorgan, Wales 

Rhondda, Wales 


2.0 

1.3 







A survey made of U.S. Indian school children 
by Paul (John E.) and Dixon in 1937 for prev- 
alence of rheumatic heart disease showed the 
same phenomenon. 



Area 


Latitude 
(degrees) 


Pre\'alenco Rheu- 
matic Heart 
Disease (percent) 


Montana and Wyoming 

Arizona (North) _ _ _ _ _ _ 


44-46 
36-37 
32-33 


4.5 
1.9 


Arizona (South) 


.5 



Of more pertinent interest are results of exam- 
inations of Indians done by the Division or Indian 
Health in 1955, when the operation of hospitals 
and health facilities was transferred from the De- 
partment of Interior to DHEW (USPHS). The 
health examination records re-analj^zed bj' Dr. 
Burch reveal a significant difference in jDrevalence 
of rheumatoid arthritis and osteoarthrosis between 
northern and southern reservations. 



Area 


Latitude 


Prevalence of 

Arthritis 

(age 15 and over), 

percent 


Montana and South Dakota 

New Mexico and Arizona 


46-48 
32-36 


27. 6 
11.2 



To test the influence of climate on arthritis it 
was thought best to select a relatively homogene- 
ous population that lived at widely difl'erent lati- 
tudes for several successive generations. The 
Blackfeet tribe (currently being surveyed) living 
in Browning, Montana at the northern border of 
U.S. at 48th parallel and the Pima tribe (to be sur- 
veyed in September, 1962), living near Phoenix, 
Arizona at 33rd parallel seemed especially suitable 
for comparison. These tribes have much in com- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



201 



mon: stable population, economic status, living 
habits, occupation, culture and racial background. 

For tlie Blackfeet survey, excellent cooperation 
and valuable guidance was obtained from the In- 
dian Health staff, both at Billings Area Office and 
the Blackfeet Reservation, as well as from the 
Area Engineering staff. The Blackfeet Tribal 
Council was receptive and helpful. We were for- 
tunate in recruiting Dr. Jolin Lawrence of Man- 
chester, England, Director of the Field Unit of 
the Empire Rheumatism Council as an NIAJID 
Visitmg Scientist for % of the duration of the 
actual survey in Montana. Dr. Lawrence had 
pioneered the northern European surveys referred 
to above and his participation in the Montana 
survey, as well as in all seven European surveys, 
should help standardize observations and criteria 
ill both continents. The jS'IAMD mobile miit con- 
sisted of the following staff members : Drs. Burch 
and O'Brien, Miss Watson (statistical clerk and 
laboratory aid) and Mr. Aldridge (radiographer). 
Six natives were employed as receptionist, chauf- 
fers, interpreters and coordinator. Two 21-foot 
trailers were used : one as reception office and ex- 
amining room and the other as x-ray and labora- 
tory unit. These trailers were obtained as surplus 
from another government agency and then re- 
paired, renovated and equipped by NIAJVID. 

The sample to be examined consisted of about 
1100 subjects randomly selected from 2,000 adult 
Indians above age 30. The survey was begim on 
October 2, 1961 and is scheduled to terminate about 
December 10, 1961. A satisfactory completion rate 
of 85% has been achieved thus far (November 30, 
1961). Results and analysis of their significance 
will be included in the 1962 annual report. (Drs. 
Burch, Bimim, O'Brien and Lawrence) 

Relatives of Patients with Sjogren's Syndrome 

In a study on the clinical features and serolog- 
ical reactions in a group of forty patients with 
Sjogren's syndrome an unexpectedly high fre- 
quency of several abnormal serologic factors was 
found. Rheimiatoid factor tested by the F II ben- 
tonite flocculation reaction was present in every 
case even though half the cases did not have rheu- 



matoid arthritis or connective tissue disease. Anti- 
nuclear factor tested by the immuno-fluorescent 
teclinique was found in 77%. Complement fixing 
antibodies reactive with normal human tissue con- 
stituents, e.g. liver, were present in 49%. Tliyro- 
globulm antibodies tested by tanned red cell tech- 
nique were found in 27%. The seinun gamma 
globulin concentration measured by paper elec- 
trophoresis was elevated in 75 % . 

Since studies on rheumatoid arthritis and on 
systemic lupus erythematosus have indicated that 
many of the above factors may be hereditary, a 
family study was undertaken to determuie what 
role genetic factors may play in the occurrence of 
clinical and serological changes observed in 
Sjogren's syndrome. 

Propositi — Fourteen patients who had been ad- 
mitted to the Clinical service of NIAMD served 
as propositi. After discharge from the hospital, 
nine of these propositi have been visited thus far at 
their homes and the closest neighbors of the same 
race, sex and approximately the same age were 
selected as "control" propositi. 

Relatives — The patients furnished a list of 281 
and the control propositi of 121 blood relatives. 
To date 84% of the parents, siblings and children, 
43% of other relatives of patients and 55% of the 
control relatives have been examined. 





Sjogren Relatives 


"Control" 

Relatives 

and 




Group I 
(first de- 
gree rel.) 


Group II 

(aU other 

rel.) 


Propositi 
Group III 


Total 


99 

83 

83.8 


192 
83 

'43. 2 


130 


Examined- 


72 


Completed (percent) 


'55.4 



I Efforts will be made to bring completion rates up to 80% during 1962. 

Only two individuals have refused to participate 
in the study. In this report the parents, siblings 
and children of the patients will be designated as 
group I, other relatives of patients as group II 
and relatives of the control propositi plus the con- 
trol propositi themselves as group III. The age 
and sex distribution was as follows : 



202 



ANNUVL REVIEW OF INTRAMURAL RESEARCH 



4 





Under 45 Years 


45 Years and Over 




Group I 


Group II 


Group III 


Group I 


Group II 


Group III 


Male - - - - 


18 
29 


32 
41 


16 
24 


18 
18 


1 
9 


15 


Female 


17 






Total.. . . - 


47 


73 


40 


36 


10 


32 







The majority of the relatives were visited in 
their homes by Dr. Burch who used a small house 
trailer equipped as a clinic. A few relatives were 
examined at tlie Clinical Center of NTH. 

A clinical history with special emphasis on rheu- 
matic complaints, ocular and oral symptoms sug- 
gestive of the sicca complex and of thyroid disease 
was obtained. The physical examination was lim- 
ited to the joints, eyes, mouth, skin and thyroid. A 
Schirmer test utilizing a strip of "Wliatman num- 
ber 41 filter paper (5 X 35 mm.) was done to meas- 
ure the rate of lacrimal secretion. Wetting of less 
than 15 mm. in five minutes was considered as 
positive. Roentgenograms of the hands and feet 
were made on all subjects, except pregnant 
females, and approximately 30 ml. of blood was 
drawn in the majority of cases for various serologi- 
cal and biochemical tests. 

The following tests were performed on the sera 
when the amount permitted : a) Rheumatoid fac- 
tor by the FII bentonite flocculation test. A dilu- 
tion of 1 :32 was considered as the minimum 
positive titer; b) Thyroglobulin antibodies as 
tested by agglutination of tanned human group O 



RliH- red blood cells coated with highly "purified" 
thyroglobulin ; c) Thyrotoxic thyroid tissue anti- 
bodies by complement- fixation reaction; d) Com- 
plement-fixing antibodies reactive with thymus 
tissue extracts; e) Gamma globulin by modified 
zinc turbidity test. Range of normal values was 
considered to be between 24 to 41 turbidity units. 
This range was estimated from the fi-equency curve 
of all determinations made in this laboratory on 
323 individuals other than patients ; f ) The pres- 
ence of antinuclear factor was determined in 29 j 
first degree relatives of propositi with Sjogren's 
syndrome and 14 relatives of control propositi. 
The immuno-fluorescent procedure of Coons and 
Kaplan was used, employing intact nuclei in mouse 
liver sections. 

Results: In comparing groups I, II, and III a 
significant difference was found in respondents 
below age 45 in the five parameters listed below. It 
is noteworthy that a consistent frequency relation- 
ship was maintained between group I (highest 
prevalence), group II (intermediate) and group 
III (lowest) in each of these five parameters. 



Prevalence of Clinical and Serological Abnormalities in Sjogren Relatives 



Parameters 



Group I 



(1° relatives), 
percent 



Group II 



(other rel.), 
percent 



Group III 



(controls), 
percent 



P value • 



2 or more diagnostic criteria (ARA) for R.A 

Reduced lacrimal secretory rate 

Abnormal circulating tissue antibodies 

Hypergammaglobulinemia 

3 or more "Sjogren" signs 



17 
29.8 
36.2 
20.9 
21.3 



5.5 
15. 1 
13.7 
11.3 

6.8 



2.5 

5.0 

12.5 

2.6 





<.01 
<.01 
<.01 
<. 05 
<.01 



I The method of Mantel and Haenszcl was used to obtain a chi square with two degrees of freedom tor evaluating simultaneously the significance of the de- 
parture from e.tpectation, sex adjusted at the 3 levels of relationship. 



These results demonstrate a definite familial ag- 
gregation of "possible" or "probable" rheumatoid 
arthritis, decreased lacrimal secretion, abnormal 



circulating antibodies to tissue constituents and 
increased gammaglobulin. The evidence cited 
above suggests that this is due to heredity rather 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



203 



tlian environment, but the exact genetic mechanism 
remains to be elucidated. (Drs. Burch, Bloch and 
Bunim) 

Program Planning for 1962 to 1964 

The most promising approaches to a better un- 
derstanding of etiology and pathogenesis of rheu- 
matoid arthritis and connective tissue diseases 
consist of the employment of immunologic and 
hujnan genetic disciplines. Well-trained, promis- 
ing, young immunologists have been recruited ; an 
immuno-chemist, an experimental immuno-path- 
ologist and one scientist who has been exploring 
the genetic (type-specific globulin groups) and 
immunological aspects of the rheumatoid factors. 
These men will join the staff of the Arthritis and 
Eheumatism Branch in 1963. 

The role of genetic factors in susceptibility to 
development of rheumatoid arthritis and other 
connective tissue diseases may be determined by 
appropriately designed family studies of propositi 
encountered in the course of our population sur- 
veys. These propositi are especially suitable since 
family units among the Blackf eet and Pima tribes 
live close together and on the same reservation for 
many generations. The prevalence of rheumatoid 
factor, clinical rheumatoid arthritis, ankylosing 
spondylitis and systemic lupus erythematosus, as 
determined to date by our unit, seems to be greater 
in the Blackf eet Indians than in the general popu- 
lation of the United States. 

It is also planned to study, in adequate numbers, 
identical and fraternal adult twins, one or both 
of whom has or had rheumatoid arthritis, an- 
kylosing spondylitis, osteorarthrosis or gout. 

Effects of Steroids, Essential Amino Acids, 
Pyridine Nucleotides and ADP on Struc- 
ture and Catalytic Activity of Glutamic 
Dehydrogenase ( GDH ) 

As reported in the 1960 Annual Report, in col- 
laboration with Dr. Gordon M. Tomkins of the 
Section on Metabolic Enzymes, it has been ob- 
served that various steroid hormones and diethyl- 
stilbestrol inhibit the glutamic dehydrogenase re- 
action by promoting the dissociation of the enzyme 
molecule into 4 subunits. We observed further 
that these subimits possessed alanine dehydro- 



genase activity. Thus, by altering the physical 
state of the enzyme molecule, both the. catalytic 
activity and substrate specificity of the enzyme 
were changed. Both these effects were antago- 
nized by ADP and DPN. This general problem 
has now been extended along several lines in order 
to confirm the basic observations, and to increase 
our knowledge of the nature and the consequence 
of the steroid-GDH interaction. 

Evidence for Change in Physical State of Enzyme 

Dr. Tomkins initiated a study of the light scat- 
tering properties of GDH which we have con- 
tinued in this laboratory. By this means, we have 
been able to confirm that the enzyme molecule is 
indeed disaggregated into subunits by diethylstil- 
bestrol and various steroids, and that this disaggre- 
gation is antagonized by ADP and L-leucine (see 
below) . It has been repeatedly borne out that a 
low concentration of DPNH, TPNH or DPN must 
be present in order for the steroids or steroid ana- 
logs to disrupt the enzyme molecule. This re- 
quirement for DPNH is distinctly different from 
that required for the catalysis of the chemical 
transformation as shown by distinctly different 
dissociation constants. It is interesting that the 
value of this constant is of the same order as that 
for the DPNH enzyme complex demonstrated by 
a study of DPNH fluorescence enhancement by 
GDH (as shown by Dr. Tomkins). Studying the 
kinetics of the disruption by diethylstilbestrol with 
this technique, it is apparent that only when the 
enzyme is completely dissociated does alanine de- 
hydrogenase activity appear, while GDH activity 
is lost much more rapidly ; i.e. only the most asso- 
ciated form of the enzyme functions as GDH, 
wloile the most dissociated form functions as ala- 
nine dehydrogenase. Other pertinent observations 
made with light scattering include the fact that all 
of the substrates, with the exception of ammonia, 
appear to have some effect on the physical prop- 
erties of the enzyme. 

Effects of L-leucine and Other Essential Amino 
Acids on Structure and Function of GDH 

It has been observed that the essential amino 
acid, L-leucine (but not D-leucine) stimulates the 
rate of oxidation of glutamate catalyzed by either 
the crystalline enzyme from beef liver or a crude 
preparation of rat liver mitochondria by as much 



204 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



as 100% . In so doing, it protects the enzyme from 
inhibition by steroids, phenanthridine and DPNH. 
Sedimentation experiments have revealed that 
leucine functions to maintain the enzyme in the 
aggregated state. The essential amino acids, iso- 
leucine and methionine, and the non-physiologic 
amino acid, norvaline, are also effective though 
somewhat less so than L-leucine. Leucinamide, 
leucylleucine, and leucylglycine were inactive. 
The amino acids, glycine, alanine, valine, lysine, 
serine, threonine, norleucine, arginine, ornithine, 
tryptophan, phenylalanine, histidine, tryosine and 
a-aminobutyrate were all ineffective, as were the 
a-keto analogs of leucine and isoleucine. Thus a 
high degree of specificity was observed for the 
leucine effect. These findings may represent a 
mechanism by which essential amino acids can 
regulate general ammo acid meatbolism. 

Dissociation hy Steroids and Other Agents 

To gain some knowledge about the structural 
requirements for the steroid inhibition of the 
GDH reaction, the effects of a large number of 
analogs of progesterone, estradiol and androstene, 
3,17-dione liave been examined. It was found 
that even minor alterations of the steroid nucleus, 
particularly those which disturb either the pla- 
narity of the molecule or increase its polarity can 
drastically reduce inhibitory potency. It is also 
interesting that the introduction of a double bond 
in the 16 position in progesterone enliances its ac- 
tivity three-fold. Thus, the inhibitory potency of 
the types of steroid tested roughly correlated with 
the degree of polarity ; those compounds that are 
least polar are most inhibitory. 

As mentioned, the presence of pyridine nucleo- 
tide is required for disaggregation of GDH to 
proceed. It has also been observed that both the 
catalytic activity of the enzyme and its respon- 
siveness to steroid inhibitors are markedly affected 
by salt concentration. The fact that certain SH 
groups in the enzyme are important in determin- 
ing whether or not steroid can control the reaction 
was reported in the 1960 Annual Report. This 
observation has now been confirmed, and the addi- 
tional observation made that parachloromercuri- 
benzoate not only can modify the inhibitory effect 
of the steroid hormones, but also can prevent 
stimulation of the enzyme by ADP and leucine ; 
thus this SH reagent appears to interfere with 



the control of the enzyme both as regards inhibi- 
tion and stimulation. 

Previous workers have shown that o-phenan- 
throline (op), a potent zinc-bmding reagent, can 
also dissociate GDH into subunits. This led to 
the proposal that zinc was responsible for main- 
taining the aggregated state of the pi'otein. Our 
findings with the steroid hormones led us to ques- 
tion whether "op" might work by some other mech- 
anism. Accordingly, we have found that phen- 
anthridine, m-phenanthroline, and phenanthrene- 
9-aldehyde, all non-chelating analogs of "op", also 
dissociate the enzyme and thus inhibit the GDH 
reaction. ADP antagonizes the action of these 
compoimds also. A study of the properties of 
these molecules may lead to a better understanding 
of the mechanisms by which the enzyme is 
disaggregated. 

A number of other amino and keto acids have 
been examined as substrates for glutamic dehy- 
drogenase, and evidence has been obtained sug- 
gesting that norvaline, a-ketovalerate, a-amino- 
butyrate, a-ketobutyrate, a-ketocaproate and 
a-ketoisocaproate serve as substrates for a form 
of the enzj'me intermediate between the fully as- 
sociated and the fully dissociated state. Thus, 
with maximum dissociation of the molecule (di- 
ethylstilbestrol at pH 8 and above) the reactions 
involving these substrates are inhibited, while 
partial dissociation (diethylstilbestrol at pH 7) 
results in stimulation. Both of these effects are 
again reversed by ADP. Thus, glutamic dehy- 
drogenase appears to have a spectrum of activi- 
ties depending on its state of aggregation. 

In summary, we have been able to confirm the 
relationship between physical structure and cata- 
lytic activity' of GDH by independent means of 
measurement. From our studies, it becomes ap- 
parent that control of this enzyme is a rather 
specific process and apparently' involves specific 
sites on the enzyme molecule wliich are concerned 
with control. This is borne out by the fact that 
there appears to be a specific binding site for 
pyridine nucleotide, which is concerned with con- 
trol, as well as the site for steroid, ADP and leu- 
cine. Wliether or not some of these sites are 
identical has not as yet been determined. 

The question of the intimate mechanism of the 
steroid effect has not been elucidated. The fact 
that the enzyme molecule can come apart rather 



fl 



I 



\\ 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



205 



easily on dilution suggested that the attractive 
forces which maintain the enzyme in the aggre- 
gated state are some sort of weak, non-covalent 
interaction. (Dr. Yielding) 

Ejfect of Steroids on PhosfliofyrwoiG Kinase 
{PEP Kinase) 

The fact that PEP kinase is so sensitive to salt, 
plus the reported observation that urea dissociates 
it into subunits, prompted us to test the effects 
of steroids. It was observed that DES and estra- 
diol were moderately good inhibitors of the reac- 
tion and, interestmgly, that this effect could be 
antagonized by high concentrations of DPN. 
Urea, deoxycholate and sodium dodecyl sulfate 
also inhibit the reaction. Examination of the 
crystalline enzyme in the analytic ultracentrif uge, 
as well as in sucrose gradient density centrifuga- 
tion, failed to reveal any evidence of dissociation 
of the enzyme molecule. Similarly, experiments 
with light scattering showed no gross change in 
the weight average molecular weight under the 
influence of diethylstilbestrol. Agar-gel electro- 
phoresis has now revealed that the inliibitory 
steroids cause a drastic change in the electrophor- 
etic properties of the enzyme, and that these 
changes are well correlated with the kinetic find- 
ings. (Drss. Yielding and liimberg) 

Metabolism of Aromatic Amino Acids and 
Homogentisic Acid in Phenylketonuria, 
Alcaptonuria and Ochronosis 

Objectives in studying patients with diseases 
associated with abnormal metabolism of the aro- 
matic amino acids, namely, alcaptonuria, phenyl- 
ketonuria, tyrosinosis and albinism, have been 
several: 1) to determine the exact nature of the 
metabolic defect in these conditions; 2) to study 
the hereditary pattern of "these diseases and, if 
possible, to develop tests which will detect the 
heterozygous state in relatives carrying the trait; 
3) to study the formation and deposition of the 
pigment derived from homogentisic acid and to 
determine how it produces the pathological 
changes in the connective tissues, particularly the 
joints (ochronotic arthritis) ; 4) to study the 
cause of ochronotic arthritis, nearly always asso- 
ciated with alcaptonuria, and 5) to attempt vari- 
ous means of treatment of these metabolic diseases. 



Phenylketonuria 

Our method for blood phenylalanine has been 
modified so that it can be used with 0.1 ml. of 
whole blood. This permits quantitative determi- 
nation of phenylalanine in the small samples of 
blood obtained by heel prick from newborn babies. 
The practical usefulness of the method has been 
tested by obtaining blood samples in duplicate 
from approximately 40 babies at the D.C. Gen- 
eral Hospital. The analyses were made without 
teclinical difficulties, indicating that the method 
is applicable for such a determination, but an un- 
expected finding was that even though none had 
phenylketonuria, approximately 10 percent of the 
babies had significantly elevated levels of blood 
phenylalanine or tyrosine, and two had elevated 
values of both amino acids. Nearly all of the 
babies with elevated levels had the so-called 
"physiological jaundice of the newborn" and we 
suspect that the elevated levels were due to the 
immaturity of the liver and the delayed develop- 
ment of the liver enzymes concerned with the 
metabolism of these amino acids. Further analy- 
ses are beuig made m premature infants and in 
older white and negro babies. 

Evidence of elevated phenylalanine levels in 
non-phenylketonuric babies is of considerable 
clinical importance, since it has been generally 
assumed that this laboratory finding alone is 
enough evidence to make the diagnosis. Obvi- 
ously other data are necessary, even in families 
with known phenylketonuria. 

Tyrosine and ascorbic acid 

It has been known for many years that guinea 
pigs and man deficient in ascorbic acid metabolize 
tyrosine incompletely when given extra amounts 
of this amino acid. Studies in our laboratory over 
the past few years have showii that vitamin C acts 
in only one of the enzymatic steps in tyrosine 
metabolism — ^the oxidation of p-hydroxy-phenyl- 
pyruvic acid to homogentisic acid. In this reac- 
tion, ascorbic acid does not function as a specific 
cofactor of the enzyme, but as a nonspecific reduc- 
ing agent which protects p-hydroxyphenylpyruvic 
acid oxidase from inhibition by its substrate. 
However, a number of other reducing agents can 
also protect the oxidase from substrate inhibition, 
such as analogues of ascorbic acid, some quinones 
and a variety of dyes, such as 2,6-dichlorophenol- 



206 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



indophenol. Furthermore, studies with purified 
enzyme preparations have indicated that ascorbic 
acid protects the oxidase indirectly by reducing 
another liver component. Evidence that ascorbic 
acid is acting indirectly in this reaction is that 
with each stage of purification of the enzyme, 
ascorbic acid becomes less and less effective in 
protecting the oxidase from inlaibition. 

Recently we have attempted to determine the 
chemical structure which is shared in common by 
the compounds able to protect the oxidase. These 
studies have been undertaken to learn more about 
the mechanism by which the enzyme is protected, 
and to give us an idea of the compound through 
which ascorbic acid acts to protect the liver enzyme 
in vivo. 

In vitro experiments have shown that coenzyme 
Qxo is the only naturally occurring compound 
which has both the proper structural requirement 
to protect the oxidase and much greater effective- 
ness in the presence of ascorbic acid. With puri- 
fied oxidase preparations, the combination of Qio 
and ascorbic acid is at least 1000 times more effec- 
tive than either ascorbic acid or Qio alone. In 
vivo evidence that coenzyme Qio participates in 
tyrosine metabolism has also been obtained. 
Frankly scorbutic guinea pigs (completely de- 
pleted of ascorbic acid) did not have their p- 
hydroxyphenylpyruvic acid oxidase protected 
When Qio was injected intraperitoneally. How- 
ever, moderately vitamin C-deficient guinea pigs 
(liver concentrations of ascorbic acid reduced to 
approximately 6 mg%) had complete protection 
of their liver pHPP oxidase when Q,io was in- 
jected. These animals moderately deficient in 
Vitamin C would have had complete inhibition of 
their oxidase if not given Qio. These in vivo sug- 
gest that the liver component through which 
ascorbic acid acts is coenzyme Qio or a closely 
related quinone. Further studies on the role of 
coenzyme Qio in the metabolism of tyrosine are 
under investigation. 

Connective tissue and ochronosis 

Our studies are completed on the distribution of 
homogentisic acid in the tissues of guinea pigs at 
various times after injection of the acid intra- 
peritoneally. Young animals were found to have 
the same unusual distribution pattern of relatively 
large concentrations in skin and cartilage and very 



little in muscle and brain, as we found earlier in 
older animals. We can therefore eliminate the 
possibility that aging of the collagen explains the 
high affinity of the connective tissues for homo- 
gentisic acid. Binding experiments to determine 
the affinity for homogentisic acid of homogenates; 
of skin, cartilage and serum albumin have shown, 
that there is no significant binding under equilib-| 
rium dialysis conditions for skin or cartilage, but 
homogentisic acid is bound to the extent of 40 per- 
cent by serum albumin. The behavior of homo- 
gentisic acid is thus very similar in its binding and 
distribution properties to gentisic acid. 

We have also developed a new specific method 
which allows us to determine small amounts of 



ofjj 
off' 



benzquinone acetic acid, the oxidized foim 
homogentisic acid, in blood and tissues. Since 
this quinone is the likely first intermediary prod- 
uct in the formation of the ochronotic pigment in 
the connective tissues, this method will be of value 
in studying the distribution and fate of homogen- 
tisic acid via this oxidative pathway. 

Alcaftonuria 

The nenal clearance of homogentisic acid in an 
alcaptonuric patient has been studied. Gentisic 
acid, a close analogue of homogentisic acid which 
is also actively secreted by the kidney tubles, does, 
not alter the renal secretion of homogentisic acid, 
nor does it cause an elevation in the plasma level 
of homogentisic acid in the alcaptonuric patient. 

We suspect that the remarkable ability of the 
kidney to secrete homogentisic acid is an impor- 
tant protective mechanism to delay the develop- 
ment of ochronosis in alcaptonuric subjects and 
that this ability may gradually become less effec- 
tive with age. Attempts to measure the capacity 
of the kidney to secrete homogentisic acid have 
been made by oral loading tests with L-tyrosine. 
Even though these resulted in establishing very 
high plasma levels of HGA, essentially complete 
renal clearance was found, even at tlie elevated 
levels. Wlaether the capacity is less in older al- 
captonuric patients is not known and should be 
determined. Neither sulfinpyrazone nor probene- 
cide had any effect on the plasma level of homogen- 
tisic acid, nor the renal clearance of HGA. 

Attempts to inhibit the formation of homogen- 
tisic acid with Antabuse (the disulfide form of 
diethyldithiocarbamate which is a very potent 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



207 



inhibitor in vitro of the enzyme which forms 
homogentisic acid) did not inhibit this pathway 
in the alcaptonuric patient. This method of treat- 
ment is of theoretical interest and we will continue 
to search for an effective agent to inliibit p-hy- 
droxyphenylpyruvic acid oxidase in vivo. 

An alcaptonuric patient was also given a few 
grams of shikimic acid to see whether this com- 
pound could be metabolized to homogentisic acid. 
Shikimic acid is an intermediate in the synthesis 
of the aromatic amino acids in bacteria and in 
other organisms able to make their own tyrosine 
and phenylalanine. Man cannot make pheny- 
lalanine (and tyrosine) directly, presumably be- 
cause of a metabolic defect somewhere along this 
pathway, although exactly where has not been de- 
termined. Alcaptonuric patients represent ideal 
subjects to study for the location of this defect, 
because nearly all of any phenylalanine formed 
would be changed to homogentisic acid. Shikimic 
acid did not yield homogentisic acid but further 
studies with other intermediate compounds are 
planned. (Drs. LaDu, Seegmiller, Zamioni, Ma- 
la wista and Jacoby) 



Gout 

Effect of Certain Uricosuric Agents on Renal 
Clearance of Uric Acid 

In an effort to gain a better understanding of 
the mechanism involved in the renal excretion of 
uric acid, we have continued studies of the effects 
of various drugs on uric acid excretion. The 
uricosuria noted in last year's report to accom- 
pany the administration of azauridine to cancer 
patients has been shown to be the result of two 
actions of the drug. The first seems to be a direct 
effect of azauridine on the kidney. Tlie second 
action is an indirect one mediated by orotic acid 
which accumulates in cells as a consequence of the 
I metabolic block in pyrimidine metabolism and is 
excreted in substantial amotmts in the kidney. 
The intravenous infusion of orotic acid as the 
sodium salt also produced a uricosuria. Certain 
similarities of chemical structure shared by these 
three compounds suggest that these compounds 
may be utilizing a common transport mechanism 
in the kidney. The necessity of administering 
these compounds intravenously limits their clini- 



cal usefulness. An enzymatic method has been 
developed for determining microgram quantities 
of orotic acid in biological fluids which will per- 
mit further studies of the mechanism involved in 
the renal excretion of orotic acid. 

The marked antagonism of uricosuric doses of 
sulfinpyrazone and of ux'icosuric doses of salicy- 
late has been investigated by determining simul- 
taneously the renal clearance of uric acid and of 
inulin. In a gouty patient receiving sulfinpyra- 
zone the urate clearance was reduced to 5% of tlie 
initial value by the intravenous administration of 
sodium salicylate without a significant change in 
glomerular filtration rate as revealed by inulin 
clearances. After the infusion was discontinued 
the urate clearance was further reduced to 0.5% 
of the initial value. These results suggest that 
addition of salicylates dissociates two aspects of 
the renal handling of uric acid in almost com- 
pletely reversing the block in tubular reabsorption 
of filtered urate induced by sulfinpyrazone and 
at the same time blocking the tubular secretion of 
urate. The net result is an almost complete 
suppression of uric acid excretion. (Drs. Seeg- 
miller and Howell) 

Acute Arthritis Induced in Gouty Subjects hy 
Intra-articular Injection of Microcrystalline 
Urates 

The way by whicli the hyperuricemia of gouty 
patients may give rise to the acute attack of arth- 
ritis has been under investigation. We have been 
able to show that the introduction of a suspension 
microcrystalline sodium urate crystals into the 
knee joint of volunteer gouty subjects gives rise to 
an inflammatory reaction, while similar injection 
of amorphous suspensions or solutions of sodium 
urate gave rise to little or no response. The pain, 
warmth, swelling and effusion resulting from the 
injections usually subsided spontaneously within 
24. hours, but in some patients was accompanied 
by typical acute gout involving joints in addition 
to those injected. Phagocytosis of urate crystals 
was observed in both the induced inflammatory 
response and spontaneous acute attacks of gout. 
The fact that urate crystals are obserA-ed in joint 
fluid of some gouty patients between attacks witli- 
out phagocytosis suggests that factors in addition 
to the simple presence of urate cr\^stals may be 
involved in the genesis and propagation of the 



208 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



acute attack of gout. (Drs. Seegmiller and 
Howell) 



Cystinosis 

Studies have been made of the presmned ab- 
normality of cystine metabolism which results in 
the deposition of cystine crystals in the tissues of 
patients with Lignac-Fanconi syndrome with 
cystinosis. The incorporation of cystine-S^^ into 
urinary sulfur compounds has been studied in three 
cystinosis patients and in three non-cystinosis 
patients. Data now available show a substan- 
tially smaller amount of S^^ in total sulfates in the 
two cystinosis patients than in the one normal sub- 
ject whose studies are now completed. Studies 
have also been made of the rate of oxidation of 
cysteine to cystine by plasma of normal and cysti- 
nosis patients without revealing a significant dif- 
ference. Likewise no substantial difference in 
activity of enzyme systems concerned with reduc- 
tion of disulfide groups has been demonstrable in 
either erythrocytes or in liver obtained post 
mortem. These include cystine reductase which 
catalyses the reduction of cystine with DPNH as 
the cofactor, cystine transhydrogenase, which re- 
duces cystine in the presence of reduced gluta- 
thione, and homocystine transhydrogenase, an en- 
zyme which reduces homocystine and which has 
previously been described in other tissue by 
Backer. (Drs. Seegmiller and Howell) 



Demonstration of Enzymatic Defect in Acatalasia 
in Human Cell Lines (in vitro) 

It has been demonstrated that the metabolic de- 
fect of acatalasia persists in tissue culture obtained 
from not only the homozygous carriers of the ab- 
normal gene but also from persons who are hetero- 
zygous for the abnormal gene. In the latter, no 
catalase activity was demonstrable in cell lines 
propagated m vitro as long as three months. Cell- 
free enzyme preparations from normal cells 
showed no inhibition of catalase activity upon 
addition of similar cell-free enzyme prepared from 
acatalasic cells. (Drs. Howell and Krooth, 
NIAID) 



Oinical Trial of Anti-rheumatic Drugs in Rlieu- 1 1 
matoid Arthritis, Psoriatic Arthritis and! 
Lupus Nephritis ■ 

Anti-folic Acid Compoimds in Psoriatic Arthritis 

This study is being conducted in collaboration! 
with Dr. Eugene Van Scott and Dr. Eobert Auer-j 
bach of the National Cancer Institute. As of thisj 
date 13 patients have been or are currently being 
studied. Eleven of the group have completed tlie 
protocol. The clinical response to amethopterin, 
administered in a series of three injections of 1 to 3 
mg/kg at 10-day intervals, is being evaluated. 
Parameters of measurement have included sedi- 
mentation rate, changes in area and degree of skin 
involvement, the joint index reflecting the tender- 
ness, pain on motion, and swelling of the joints, 
and morning stiffness. The results of the first 11 
patients, although not statistically significant in 
this low nmnber, have been encouraging. It is 
anticipated that between 20 and 30 more patients 
will be required for this study. 

Side effects observed have been mild. The most 
prevalent complaint has been nausea and anorexia 
for the first 2-4 days following injection of the 
anti-folic compound. One patient developed oral 
ulceration and another alopecia. One death has 
occurred in this group two months following the 
final dose of the anti-folic compound. This death 
was due to cerebral thrombosis and was believed 
unrelated to the medication. Seven patients have 
shown a recurrence of psoriasis and arthritic 
symptoms between one and three months follow- 
ing the last injection. Four of these 7 have re- 
ceived a second course and have shown satisfactory 
improvement a second time. 

Hydroxychloroquine 

A study of the efficacy of hydroxycliloroquine in 
treatment of rheiunatoid arthritis has continued 
under the guidance of the American Eheumatisra 
Association's Committee on Cooperating Clinics, 
The work on this project has been candied out at 
the D.C. General Hospital in the Georgetown 
Rheumatology Service Clinic. The current trial 
(trial II) has been completed and our Institute's! I 
participation was to the extent of 14 patients. 
The analysis of this double-blind study is as yet 
not complete. 



"NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



209 



DexametJi^isone and Prednisone 

The long-tenn foUo^vup study of these com- 
pounds has continued with the re-eyaluation of the 
patients receiving this medication. Posterior sub- 
capsular cataracts (PSC) , reported last year, have 
been found in additional steroid-treated patients. 
In all, 95 rheumatic disease patients have been 
evaluated. Of these, 72 patients have received 
corticosteroid therapy. Thirty of these 72 had 
PSC on examination. This evaluation, an exten- 
sion of the study reported one year ago, continued 
to show the same relationsliip of the development 
of cataracts to both the dosage and duration of 
corticosteroid administration. 

6a. Fluorotriamcinolone 

Metabolic balance studies of a total of 3 patients 
have been completed, employing 20 mgm. of tliis 
corticosteroid daily (Drs. Whedon and Lutwak). 
Based on the observations of the first 2 patients 
there is some suggestion that this material is capa- 
ble of causing a positive calcium balance. The 
result of the third study is as yet undetermined. 
Five patients have been previously studied in our 
Institute, receiving short-term course of 6a 
fluorotriamcinolone. The antirheumatic poten- 
cy of this compoimd was found to be midway be- 
tween that of dexamethasone and prednisone — 
% to 1^ the potency of dexamethasone and I14 to 3 
times the potency of prednisone. A study of the 
pituitary inhibiting potency was made on one pa- 
tient, and it was found that 4 mgm. of 6a fluorotri- 
amcinolone daily was inadequate to completely 
suppress the pituitary as measured by plasma 
hydrocortisone levels. This would suggest that 
the compound is somewhat less potent as a pitui- 
tary suppressant than is dexamethasone. 

A long-term trial of the anti-inflammatory effect 
of this steroid has been instituted at the Eheuma- 
tology Clinic at the D. C. General Hospital. It is 
planned that 10 patients will participate in this 
trial, 3 of whom have already received the com- 
pound from 4r-5 months each. The dosage em- 
ployed has been from 6-10 mgm. daily. As of 
this date no significant side effects have been ob- 
served. These patients are receiving periodic 
radiologic examination to assess the degree of 
osteoporosis while receiving this drug. 



High Dosage Corticosteroid TTiera-py in Lupus 
Nephritis 
The efficacy of high doses of corticosteroids 
(equivalent to 50 mgm. of prednisone) in the 
treatment of the renal lesion of lupus has been 
reported by Robert Kark and co-workers at the 
University of Illinois. Their data show the sig- 
nificant reduction in mortality among patients 
treated in tliis fashion as compared with the 100% 
mortality in their control series. Their data also 
show regression of the renal lesions, as observed 
by serial biopsies, during liigh dosage corticoster- 
oid treatment. We have observed at the Clinical 
Center four patients with systemic lupus erythe- 
matosus and biopsy evidence of nephritis. One 
patient was treated xoithout corticosteroid therapy 
and two other patients have been treated with 
prednisone, 50 mgm. daily. In all tliree of these 
a repeat biopsy, performed three to four months 
later, showed regression of the renal lesion. The 
fourth patient has not as yet been treated, al- 
though the high dose regimen will be employed 
in tliis case. It is important that no effects have 
been observed in these patients at high dosage, 
other than severe Cushingoid facies and marked 
weight gain have been noted. (Drs. Black, 
Bunim, Kimberg, Wohl, Bitensky and Howell) 



GASTROENTEROLOGY UNIT 

Whipple's Disease 

Therapy 

In all three patients with Wliipple's disease 
studied at NIAMD it has been observed, as was 
previously reported by others, that patients may 
fail to respond satisfactorily to treatment with 
corticosteroids but respond dramatically when 
antibiotics are added. A remission of the disease 
is thus induced. 

Hypoalbuminemia 

With Dr. Thomas Waldmann of NCI we have 
demonstrated for the first time that the hypoal- 
buminemia of this disease is attributable, at least 
in some instances, to enteric leakage of albumin 



210 



ANNUAL REVIEW OF INTRAMXJRAL RESEARCH 



and that this exudative enteropathy reverses it- 
self when therapy induces a remission. 

Electron Microscopic Studies 

With Dr. William G. Banfield (NCI) ^ye have 
studied the liistological changes in intestinal 
biopsy specimens from patients with "Wliipple's 
disease using the electron microscope. We have 
found, as have two other groups recently, that the 
tissues contain unidentified bodies the size of small 
bacteria or large viruses. We have extended the 
observations made by others to show that the 
number of these bodies in the tissue apjDears to 
parallel the patient's clinical state. Attempts to 
culture these bodies using methods suitable for 
detecting bacteria or viruses have been unsuccess- 
ful to the present time. We have not yet had the 
opportunity to obtain tissue from a lymph node of 
a patient with Wliipple's disease. Such tissue 
should offer greater chance to culture an organism 
if that is what these bodies are. 

PAS Stained Bodies in Macrophages 

With Dr. Samuel Spicer of NIAMD we have 
studied the histochemical appearance of the ma- 
terial that is present in tissue macrophages of 
patients with Whipple's disease and is detectable 
by use of the periodic acid — Schiff stain. Dr. 
Spicer believes that the PAS positive material is 
different from normal intestmal mucus, that it is 
not a sialomucin and that it is not a sulfated 
mucopolysaccharide. (Dr. Laster) 

Hypogaimnaglobulinemia 

Together with Dr. Waldmann of NCI we have 
completed a study that has demonstrated the fol- 
lowing: Patients that present with hypogamma- 
globulinemia as their major disturbance have, in 
a high percentage of instances, an associated hypo- 
albuminemia. Tliis has not been appreciated 
heretofore. We have studied six such patients, 
many of them having concomitant intestinal dis- 
eases, and have found that the hypoalbuminemia 
can be due to 1) impaired synthesis, 2) abnormal 
loss of albumin through the gastrointestinal tract, 
and 3) a combination of these two mechanisms. 
We have also found that when therapy is capable 
of improving the patient's condition to the extent 
of brinffing the albumin levels back to normal in 



those patients with excessive enteric leakage of i 
albumin, the gammaglobulin remains abnormally ' | 
low and does not return to normal in parallel with 
the albumin. (Dr. Laster) 

Hypolipidemia 

In the last Annual Report we described a con- 
dition known as A-beta-lipoproteinemia. We 
have since acquired a second patient with this 
extremely rare disorder and, together with Dr. 
John Bieri of NIAMD, have shown that in both 
patients there is a striking deficiency of vita- 
min E. 

We are also studying the genetics of this dis- 
order and have found that although one of our 
patients has no beta-lipoprotein, his mother, f atlier 
and sister have normal levels of this serum con- 
stituent. This finding contrasts with that re- 
ported from England in which each of the parents 
of a propositus had half normal levels of serum 
beta-lipoprotein. These observations leave the 
question of the genetic aspects of the disorder 
somewhat confused at the present time. (Dr. 
Laster) 

Inborn Errors of Metabolism 

In our previous annual report we indicated 
our intention to study the biochemistry of the 
human intestinal mucosa and to utilize this readily 
available tissue to study derangements of bio- 
chemistry in man. In collaboration with Di-s. 
Topper and Segal of NIAMD, we have shown 
that intestinal mucosa from a patient with galac- 
tosemia has an extremely low capacity to meta- 
bolize galactose 1-C^^ in contrast to mucosa from 
non-galactosemic patients. Thus, the intestinal 
mucosa is an excellent tissue for studying other 
inborn errors of metabolism, not only because of 
its availability by biopsy methods (and we have 
done about 250 intestinal biopsies since our unit 
has been started), but also because a wide variety 
of pathways of metabolism can be detected in 
this extremely active tissue. 

Extending this further in collaboration with 11 
Drs. Field and Williams of NIAMD, we have "' 
studied a sibling and the two parents of a patient 
with von Giei-ke's disease and have found that 
the brother and mother had levels of intestinal 



II 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



2L 



glucose-6-phospliotase that were comparable to 
those obtained in studies of mucosa from control 
subjects, but that the activity of the mucosa from 
the patient's father was half normal. We chose 
not to biopsy the patient himself because of a 
bleeding tendency (not uncommon in this dis- 
ease). Since the intestinal biopsy is much safer 
than a liver biopsy, this observation extends our 
diagnostic armamentarium in relation to glycogen 
storage diseases. 

We are in the process of developing methods to 
utilize this tissue to study other inborn errors of 
metabolism involving pentoses, purines and 
pyrimidines. (Dr. Laster) 

Biliverdin Reductase 

We found that crystalline albumin added to a 
pure preparation of this enzyme caused a marked 
stimulation of biliverdin reduction by DPNH but 
not by TPNH. This observation is being investi- 
gated further. The only incomplete area in this 
study is our inability to obtaia satisfactory values 
for the stoichiometry of the reaction. This may 
be due to the fact that we do not have sufficiently 
pure biliverdin. This compound is difficult to 
prepare and we have recently come upon a new 
synthesis which may allow us to bring these 
studies to a satisfactory conclusion. (Drs. Single- 
ton and Laster) 

CLINICAL EINDOCRINOLOGY BRANCH 

Carbohydrate Metabolism 

Ghicose 

Last year a method was devised for analyzing 
the extent of glucose metabolism by the hexose 
monoj)hosphate oxidative pathway (HMP) in in- 
tact human subjects. Detailed kinetic analyses 
for normal man were presented. The work has 
now been extended to patients with hypo- and 
hyperthyroidism. Interestingly enough, the pro- 
portion of the overall glucose metabolism attrib- 
utable to this "shunt" pathway was decreased in 
both types of patients. The absolute quantity of 
glucose traversing the HMP pathway, however, 
was increased in hyperthyroidism and decreased 
in hypothyroidism due to the marked correspond- 



ing changes in overall glucose oxidation. As a 
part of these studies, a new and simplified method 
was developed for determining the specific activity 
of C" gluconate by employing direct liquid scintil- 
lation counting of filter paper segments. (Dr. 
Segal) 

The study of glucose metabolism in isolated tis- 
sues — particularly the endocrme glands — has con- 
tinued. The in vitro effect of thyroid stimulating 
hormone (TSH) on thyroid slices has been exam- 
ined further. A more sensitive assay for TPN 
and TPNH based on oxidation of 6-phosphogluco- 
nate-l-C" was developed, and provided confirma- 
tion of the previous finding that TSH causes an 
increase in TPN. TPNH fell somewhat, but not 
enough to accomit for the rise in TPN. Attempts 
to obtain similar effects with thyroid homogenates 
were again unsuccessful. Epinephrine and sero- 
tonin, on the other hand, stimulate glucose-1-C^* 
oxidation in thyroid mitochondria and micro- 
somes, apparently through increasing TPNH oxi- 
dation. Wliereas epinephrine acts catalytically 
on thyroid slices, serotoniii does not. 

The effect of TSH on glucose oxidation in bovine 
thyroid slices has been employed as an assay for 
TSH in human plasma. The method is sensitive 
to .014 noilliunits of TSH, and gives a linear re- 
sponse over the range .014 to 0.5 millimiits. Nor- 
mal human plasma contains 1-10 milliunits/100 
ml. (Drs. Field & Pastan) 

In bovine anterior pituitary slices, the aldehydes 
corresponding to epinephrine and serotonin were 
as active as the amines in stimulating glucose ox- 
idation, but the acid and alcohol analogues were 
without effect. The catechol amines and their al- 
dehydes were effective in homogenates fortified) 
with TPNH, suggesting an action on TPNH oxi- 
dation, and tliis effect was localized to the super- 
natant fraction. A number of tryptamine and 
phenylethylamine derivatives related to epineph- 
rine and serotonin also increased oxidation of 
glucose-1-C" by pituitary slices and the effect 
could be blocked by monoamine oxidase inhibitors. 
(Drs. Field & Barondes) 

The various zones of the bovine adrenal cortex 
were examined. Glucose-1-C" oxidation and glu- 
cose uptake were higher in the zona glomerulosa 
compared to the zona fasciculata, whereas phos- 
phorylase was higher in the zona fasciculata. 



643351—62- 



-15 



212 



ANlSrUAL REVIEW OF rNTKAMTJRAL RESEARCH 



ACTH was found to stimulate glucose-1-C" and. 
glucose-6-C" oxidation in adrenals taken from 
hypopliysectomized rats, but was ineffective in 
normal rat or bovine adrenal slices. Epiiaeplirine 
and serotonin, however, stimulated glucose oxida- 
tion in normal adrenals. ( Drs. Field & Williams) 

Phospliorylase activity was also studied in the 
corpora lutea of bovine ovaries. This activity was 
stimulated by chorionic gonadotropin as well as 
by growth hormone, but pituitary FSH, LH, TSH 
and ACTH were without effect. Thyroid and tes- 
ticular phospliorylase were not affected by the cor- 
responding trophic honnones, but chorionic gonad- 
otrophin did increase phospliorylase activity in 
dog liver slices. Corpus luteum phosphorylase 
could also be increased by epineplirine, but was de- 
creased glucagon. 

Glucose oxidation was studied in rat salivary 
glands. Although this was stimulated by acetyl- 
choline, epinephrine, serotonin and histamine, 
atropine inhibited only the acetylcholine effect, 
antihistamines only that of histamine, and mono- 
amine oxidase inhibitor affected only the catechol 
amines. TSH and Nal had no effect on glucose 
oxidation — an interesting observation since sali- 
vary tissue actively concentrates iodide. (Field) 

It is hoped that these studies will lead to a bet- 
ter understanding of the functioning of the endo- 
crine glands. 

Hypoglycemia 

Hypoglycemia has been induced in normal hu- 
man subjects by the administration of alcohol fol- 
lowing a 48 hour fast. Tliis effect, which had been 
noted clinically, is apparently not due to increased 
insulin release since there is no concomitant in- 
crease in plasma unesterified fatty acids. 

The hypoglycemic effect of L-leucine in patients 
with islet cell tumors has been under investigation. 
Studies with isolated rat diaphragm showed no 
effect on glucose uptake, with or without added 
insulin. 

In patients with leukemia, an artifactual hypo- 
glycemia has been shown to arise from glycolysis 
by leukocytes after blood withdrawal. Methods 
have been devised to overcome this artifact. (Dr. 
Field) 



Insulin and insulin antagonists 

The effect of insulin in preventing K* and water 
loss from isolated perfused rat liver has received 
further study. A more closeh- controlled, paired 
perfusion technique has been developed, leading to 
the new observation that inhibition of K+ transfer 
is accompanied by inhibition of glucose produc- 
tion. The time courses of both effects were similar, I 
with a maximum at 90 minutes. Urea production 
was also reduced, but the time course was different. 
Gluconeogenesis was studied in livers depleted of 
glycogen, and it was shown to be increased by 
glucagon and decreased by insulin. This effect of 
insulin, however, contributed only 15% of the total 
insulin effect on glycogen-laden liver. It appears, 
tlierefore, that the major effect of insulin in the 
perfused liver is on inhibition of glycogenolysis. 
The relationship of this effect to that on K* and 
water are under investigation. (Drs. ilortimore 
& Wetzel) 

Further studies on the metabohsm of I"^-labeled 
insulin by the isolated perfused rat liver have 
shown that neither ACTH nor glucagon affect in- 
sulin degradation. This is taken to indicate that 
the degradation of insulin is specific and not shared 
by other proteins. As part of tliis study, it has 
been shown that labeled insulin is heterogeneous 
with respect to rate of degradation. A fraction 
high in diiodotyrosine content is more slowly de- 
graded, and is presumed to be less active metaboli- 
cally. (Drs. Mortimore & Tietze) 

The plasma insulin antagonists obtained during 
different clinical situations have been shown to 
alter the insulin effect on adipose tissue and muscle 
similarly. In a study of further cases of chronic 
insulin resistance treated with adrenal steroids, 
there was a reduction of required insulin dosage 
as well as insulin antagonist in all. Plasma in- 
sulin binding capacity was also reduced in 4 of 
5 cases, and insulin-I^^^ disappeared more rapidly 
than normal from the plasma of 1 of 2 cases. 
Plasma from uncontrolled diabetic patients was I 
found to inhibit glucose uptake by dog retina. Al- 
though this effect was shared by plasma from non- 
diabetics with renal failure, control of the diabetes 
resulted in a disappearance of the inhibitory fac- 
tor. This factor was shown to be non-dialyzable. 



I 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



213 



The studies indicate the presence of a varity of in- 
hibitory agents which are variously affected by 
steroids or other treatment. (Drs. Field & Keen) 

Glycogen storage disease 

The assay of j)hosphorylase in leukocytes has 
been applied to several types of glycogen storage 
disease. It is low only in cases with deficient he- 
patic phosphorylase. In one family study, the 
patient's mother had a leukocyte phosphorylase 
level about 50% of normal, while the father's was 
normal. 

In patients with glucose-6-phosphate deficien- 
cies, jejunal mucosa obtained by biopsy has been 
studied. This enzyme was normal in the mother 
and in a sibling of the patient, but the father had 
a 50% reduction from the normal. The assay in 
intestinal mucosa was found to be relatively spe- 
cific since only a small amount of phosphate was 
released from ^-glycerol phosphate. The increased 
glucose-6-phosphate concentrations reported in 
erythrocytes and plasma of heterozygotes could 
not be confirmed. (Drs. Field & Williams) 

Galactose and galactosemia 

Metabolism of C"-labeled galactose was studied 
in 8 galactosemic patients ranging in age from 
6 to 30 years. All had the typical cliuical disease 
in infancy and all had absent galactose-1-phos- 
phate uridyl transferase in their erythrocytes. 
Nevertheless, 2 patients were able to metabolize 
galactose to CO2 in nearly normal fashion. Thus, 
a subgroup of the disease has been discovered in 
which pathways of galactose metabolism exist in 
tissues other than red blood cells. 

Factors regulating galactose metabolism have 
also been examined in erythrocytes from normal 
and heterozygous individuals, and in mixtures of 
normal and galactosemic erythrocytes. It was 
found that even in the presence of a profotmd 
depression in galactose oxidation, the stimulation 
of HDP galactose epimerization by a variety of 
methods could result in a considerable stimulation 
of galactose metabolism. Thus it appears that the 
transferase enzyme does not function maximally 
even when its level imposes a profound limitation 
on galactose oxidation. (Dr. Segal) 



Amino Acid Transport and Protein Synthesis 

The occurrence of aminoaciduria in galacto- 
semia stimulated an interest in the mechanism of 
aminoaciduria, and studies with rat kidney cortex 
was undertaken. An energy dependent concentra- 
tion of ammoisobutyric acid and several C^*- 
labeled amino acids was demonstrated in tubular 
cells. The energy source has not been identified, 
but is not glucose. Kinetic analysis indicated that 
the data best fit a 2 compartment system, and the 
rate constants for influx and efflux have been 
calculated. 

This technique has been ax^plied to several ex- 
perimental situations. Maleic acid, which induces 
ammoaciduria in animals, has been shown to in- 
hibit amino acid accumulation by rat kidney cor- 
tex slices. This results from an increased rate of 
efflux, whereas influx is imaltered. Phlorizin, 
which inhibits penetration of glucose and other 
sugars into certain cells, was found paradoxically 
to increase amino acid accumulation by rat kidney 
cortex. This was due to a decrease in efflux, the 
rate of influx again remaining unaffected. 
Growth hormone, which increases accumulation of 
a-aminoisobutyric acid in rat diaphragm, had no 
effect on the rat kidney preparations. In rats 
harboring pituitary tumors producing growth 
hormone, prolactin and ACTH, however, amino 
acid accumulation by kidney slices was increased. 
The effect was shown to be dependent on adrenal 
secretion. Other studies have demonstrated com- 
petitive inhibition of lysine accumulation in the 
presence of ornitliine and arginine. 

Shnilar work has been begun utilizing thyroid 
slices. Preliminary observations indicate that 
TSH stimulates amino acid transport into this 
gland. (Drs. Segal & Rosenberg) 

The effect of growth hormone on incorporation 
of C"-leucine into protein of rat xyphoid process 
was studied. No in vitro effect of growth hormone 
was detected, but prior administration of the 
hormone to hypophysectomized animals had a 
stimulatory effect. Oxidation of glucose-1-C^* 
in cartilage was, however, stimulated by in vitro 
growth hormone. (Drs. Williams & Keen) 



( 



214 



AISnsrUAL REVIEW OF INTRAMURAL RESEARCH 



^ 



Biochemistry of the Thyroid 

Iodide transport 

Studies of the iodide concentrating mechanism 
of the thyroid gland has continued with a detailed 
comparison of univalent anions other than iodide 
which are concentrated by thyroid tissue or which 
inhibit iodide "trapping". The concentration of 
TCO4" and EeOr require cellular integrity, meta- 
bolic energy and K+, as in the case of iodide, but 
the Km values are 3 x lO-'M and 1 x 10-^M, re- 
spectively, as compared to 3 x 10"^M for iodide. 
Their transport is inhibited by CIO4-, SON" and 
BF4-, which also inhibit iodide. The K^ values, 
or Ki values for a series of inhibitors, were shown 
to bear an inverse linear relationship to the partial 
molal ionic volume ; — i.e., the larger the ionic vol- 
mne, the greater the "aiSnity" for the thyroid 
transport mechanism. Such a correlation did not 
exist with crystal radii or limiting ionic conduct- 
ances. 

Preliminary studies have shown the presence of 
a ouabain-sensitive K+-dependent ATPase in thy- 
roid tissue. Correlation of this with iodide trans- 
port is under study. (Dr. Wolff) 

lodoproteins 

The subf ractions of beef thyroglobulin obtained 
by DEAE-cellulose chromatography have been 
the subject of continued study. They were found 
to be antigenically identical and to have the same 
neutral sugar content. They differed strikmgly, 
however, in sensitivity to disaggregation by heat 
at pH 9.5. This correlated also with a difference 
in iodoamino acid composition, determined by 
spectrophotometric titration. The more stable 
fraction, which was retained more strongly on 
DEAE cellulose, contained 2.3 times as much 
iodine as the more labile fraction. The difference 
was mainly in diiodotyrosine, and some in thy- 
roxine, but the monoiodotyrosine content did not 
vary. The differences probably reflect thyroglob- 
ulin synthesized at different periods of time and 
stored in the thyroid follicles. (Dr. Robbins) 

The spectrophotometric procedure for iodo- 
amino acid analysis employed above can be ex- 
pected to have wide utility. It enables their 
determination in as little as 3 mg of intact pro- 
tein. It has also been used in the study of thyro- 
globulin iodinated in vitro in various media. 



Iodine introduced in the absence of urea is f oundj 
mainly in diiodotyrosine, whereas in 8M urea, al 
rise in monoiodotyrosine at low levels of iodina-l 
tion is observed. The formation of thyroxine is, 
the same under both conditions, but no thjToxmel 
is formed by the iodination of a trypsin digest of 
thyroglobulin. It appears that the secondary and, 
tertiary structure of thyroglobulin influence the 
pattern of iodination, and that the primaiy struc- 
ture is required for thyz-oxine synthesis. (Dr. 
Edelhoch) 

Study of the antigenic (i.e., antibody-combin- 
ing) properties of thyroglobulin has continued 
Short-time tryptic digestion produces several 
components with precipitating activity, but these 
disappear after prolonged digestion. These di- 
gests, however, still contain antigen as shown by 
inhibition of precipitation and passive cutaneous 
anaphylaxis. The active fragments vary in size 
from an average molecular weight of 700 to 8000, 
but the former give only limited inhibition of| 
precipitation. Phj'sicochemical studies suggest* 
that the antigenic activity is related only to the 
primary structure of the peptides. (Drs. Edel- 
hoch & Metzger) 



Thyroxine and iodotyrosine synthesis 

The study of the in vitro thyroxine (T.,) syn- 
thesis in which 3,5" diiodophenylpyruvic acid 
(DIHPPA) is coupled to diiodotyrosine (DIT) 
has been continued. In an attempt to further the 
analogy to biological thyroxine formation, the 
DIT peptides glycyl-L-DIT and L-DIT-glycine 
were prepared. Both reacted with DIHPPA to 
form the corresponding peptides of T4. Other 
DIT analogues also were effective. For example, 
diiodophloretic acid (desamino-DIT) plus 
DIHPPA gave tetraiodothyropropionic acid, and 
monoiodotyrosine (MIT) gave 3,3',5'-triiodo- 
thyronine. In all cases, the yields were from 10 
to 18%. 

A simple method for preparing DIHPPA was 
found to be by the use of L-amino acid oxidase (in 
rattlesnake A^enom) and DIT. Conditions were 
worked out whereby thyroxine in 10-15% yield 
could be synthesized in a one step procedure by 
the use of snake venom and DIT. This repre- 
sents a great simplification over the usual methods 
of thyroxine synthesis. 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



215 



The foregoing reactions, after modifications al- 
lowing for the use of microgram or milligram 
quantities of reactants, were applied to synthesize 
various types of labeled thyroxine. Since DIT 
forms the inner ring and side chain of T4, and 
DIHPPA forms the phenolic ring, the use of ap- 
propriately labeled starting material has led to 
the preparation of Ti"!^^^ labeled in the 3,5 or 3',5' 
positions and TrC" labeled in the phenolic ring, 
or in the inner ring and side chain. By using the 
L-amino acid oxidase procedure with DIT-I"^ or 
DIT-C", the formation of uniformly labeled T4 
has been obtained. These compounds will be ex- 
tremely valuable for studies of thyroxine metab- 
olism. (Drs. Cahnmann & Shiba) 

Thyroxine transport in Mood 

A comparative study of thyroxine-protein inter- 
action in the sera of 31 animal species, including 
mammals, reptiles, amphibians, birds and fish, was 
completed in collaboration with Drs. Farer and 
Blumberg (Epidemiology and Biometry Branch, 
NIAMD) . Only primates had thyroxine-binding 
protein patterns closely resembling those in man, 
a finding of significance in studies of thyroxine 
metabolism in animals. These findmgs also may 
be useful in taxonomic studies. (Dr. Robbins) 
*In some animals with very low serum binding 
of thyroxme, as well as in certain human diseases, 
one zone of thyroxine in electrophoretic patterns 
was shown to be an artifact arising from dissocia- 
tion of thyroxine and subsequent chromatography 
of the free thyroxine resulting from fluid flow on 
the paper strip. 

An investigation of thyroxine-binding proteins 
in early human fetuses was carried out with sera 
! collected while Dr. Eobbins was a visiting scien- 
tist in Copenhagen, Denmark. Unlike the rabbit, 
the human fetus had thyroxine-binding proteins 
qualitatively identical to the adult, although dif- 
fering in quantity. An interesting incidental 
finding was the presence in early fetal life of a 
post albumin protein in large quantity which is 
not present in adult serum or even in more mature 
fetuses. Further study of this proteiii awaits pro- 
curement of more fetal serum. (Drs. Eobbins & 
Andreoli) 

A study of the kinetics of thyroxine-binding by 
serum proteins was undertaken in collaboration 
with Dr. M. Berman (Biomathematics Office). 



In the initial studies, a rapid dialysis technique 
was developed based on dialysis from a thin layer. 
Free thyroxine dialyzed with a half time of 5 
minutes and meaningful measurements could be 
made at 2 minutes after adding thyroxine to 
serum albimiin or whole human serum. At 25 °C, 
pH 7.4, and very low protein concentrations, the 
reaction was complete in this interval. More 
rapid measurements have now been obtamed with 
a spectrophotofluorimetric teclinique, and prelimi- 
nary results are encouraging. The procedure is 
of intrinsic interest since rates of tliis type have 
not heretofore been measured. In addition, the 
rates have a bearing on problems of thyroxine 
metabolism. (Drs. Robbins & Rail) 

Previous studies of the kinetics of iodine metab- 
olism in man have revealed discrepancies be- 
tween assmned synthetic and metabolic mecha- 
nisms and the observed kinetics. Preparatory to 
an investigation of these matters, methods are 
under development for the detection of small 
quantities of labeled iodoamino acids in blood. 
Chromatography on a strong anion exchange resin 
has led to satisfactory separation of IIIT, DIT, 
iodide and the thyronines from as much as 25 ml 
of blood. Methods for separating T4 and T3 are 
under study. (Dr. Lewallen) 

Action of thyroxine on isolated systems 

The inhibitory effect of thyroxine on crystalline 
glutamic dehydrogenase (GDH), which appar- 
ently results from dissociation of the enzyme into 
subunits, has been investigated further. Dissocia- 
tion and enzyme inhibition was also produced by 
SCN, but this action was not reversed by ADP, 
in contrast to the finding with T4. The molecular 
sites of the various actions have been under study. 
Since T4 inhibits noncompetitively with respect 
to TPIST and competitively with respect to ADP, 
it was concluded that T4 inhibition is due to inter- 
action at the "activating" site, not at the "active" 
site. T3 interferes equally with fluorescence en- 
hancement of DPNH and TPNH suggesting a 
third site on the enzyme that is kinetically inactive 
but which binds these cofactors. T4 was foimd to 
inhibit metabolism of norvaline, a-ketovalerate, 
a-ketoisovalerate and a-ketobutyrate by GDH. In 
contrast, pyruvate reduction was first stimulated 
by T4, but was inhibited by liigher levels. ADP 
inhibited the metabolism of these keto acids, in 



216 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



contrast to its stimulatory effect on glutamate and 
a-ketoglutarate metabolism. (Dr. Wolff) 

Tlie effect of thyroxine on the swelling of iso- 
lated mitochondria has been mider study in col- 
laboration with Dr. E. Michel and O. Michel at 
the College de France. The swelling produced by 
10"'M T4 is accompanied by negligible (<2%) 
metabolism of T4, indicating a lack of correlation 
between these phenomena. Although ATP in- 
hibits the swelling effect of T4, it has no effect on 
binding of T4 by mitochondria, so binding and 
swelling also are without correlation. Iodine 
(I2) causes mitochondrial swelling at a dose of 
~5 X lO'^'M, and this effect is similar to that of 
T4 in a number of ways. (Dr. Rail) 

Dosimetry of P^' radiation 

A general mathematical approach to the prob- 
lem of radiation dosimetry to the bone marrow by 
I^^^ was developed. Calculated doses in a patient 
with metastatic thyroid carcinoma compared to 
reported cases of exposure to whole body x-ray 
in single exposures indicated that comparable 
hematologic effects were produced by larger calcu- 
lated doses in the former. This discrepancy was 
attributed to: (1) recovery from radiation effect 
during the relatively prolonged exposure to I"^ 
radiation, (2) absorption of ^S-ray energy by bone 
trabeculae and (3) inequality of blood and bone 
marrow isotope concentration. (Dr. Lewallen) 

Physical Chemistry of Proteins 

A study of the molecular properties of serum 
gamma globulin was pursued in collaboration 
with Dr. E. Steiner, Naval Medical Eesearch In- 
stitute. Detergents and urea produced more pro- 
found structural modifications than did acid or 
base. The effect of alkali was seen even after 
extensive unfolding by detergent or urea. Fluo- 
rescence polarization was used as an indicator of 
configurational changes in addition to the more 
conventional methods, and was found to be a more 
sensitive indicator of such changes. A study of 
the denaturation kinetics indicated heterogeneity 
in y-globulin, and it was interesting that a highly 
purified antithyroglobulin antibody showed a de- 
gree of heterogeneity similar to that of unfrac- 
tioned y-globulm. (Drs. Edelhock & Metzger) 



METABOLIC DISEASES BRANCH 
Mineral Metabolism 

Osteoporosis 

Metabolic studies from this Branch continue to 
produce evidence of the important relationship of 
dietary calcium intake to the pathogenesis and 
possible therapy of post-menopausal and senile 
osteoporosis, a disorder of thuming bones which 
by recent surveys has been shown to affect 26% 
of males and 29% of females over the age of 50 
years. As the result of the work of this labora- 
tory in conjunction with two English investiga- 
tors, it is gradually being realized that the 
long-held previous concept of the pathogenesis of 
osteoporosis (solely impaired matrix formation re- 
sulting from hormonal imbalance) is inadequate. 
The idea of multiple etiologic factors affecting 
mineral utilization is gradually being more widely 
appreciated. 

A. Effect of mcreased dietary intake of calcium 
on calcium balance. To date eleven patients with 
post-menopausal or idiopathic osteoporosis have 
been studied at several different calcium intake 
levels, balance studies being carried out for at least 
30 days at each level. Ten of these patients 
showed increased retention of calcium up to an 
intake of about 800 mgm. per day. In six pa- 
tients, the dietary calcium has been subsequently 
increased to as high as 2400 mgm. a day; three 
continued to show increasing retention of calcium 
at each higher intake level ; one patient of the six 
showed improved calcium retention at an intake 
of 1600 mgm. a da}- but not above this level ; and 
two actually showed more negative balance with 
increasing dietary calcium greater than 800 mgm. 
a day. The last two have been shown to have in- 
termittent steatorrhea, and the intestinal absorp- 
tive defect for calcium is under further study. 
Two patients who showed retention of calcium at 
Hgh intake levels have been re-examined after 
several years at the increased level and continue 
to demonstrate positive balances. 

B. Effect of dietaiy phosphate on mineral re- 
tention — preliminary results. Since the molar 
ratio in which calcium and phospliorus are re- 
tained in bone is well known (1.5), the relation- 
sliips between the relative amounts of these ele- 



I 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



217 



ments actually retained in the balance studies to 
the amoimts presented in the diet was considered to 
be of interest and possible importance. This anal- 
ysis was made possible by the fact that in most of 
the studies the phosphorus intake was changed rel- 
atively Httle as the calcium intake was successively 
raised. At a dietary molar ratio of 0.4, the re- 
tention ratio (calculated from calcium and 
phosplirous balances) was to the order of 0.8 (with 
a correlation coefficient, r, of 0.67) ; at an intake 
ratio of 0.8, retention ratio was 1.5 (r, 0.98) ; at 
intake ratio of 1.2, retention ratio was 7.8 (r, 0.18) . 
The intake ratio of 0.8 was found in our studies at 
1600 mgm. per day of calcium intake. These re- 
sults suggest that (a) the calcium being retained 
is, m fact, in bone; (b), an optimal dietary ratio 
of calcium to phosphorus may be essential for best 
absorption and utilization of these two elements. 
C. Effect of vitamin D on calcium balance. 
Moderate doses of vitamin D have been added to 
the high dietary calcium regimen in five studies 
on four patients with osteoporosis. In four of 
these studies, much more positive calcium balance 
was achieved. The full significance of these re- 
sults is not clear, but they suggest that Adtamin D, 
although hitherto believed to be closely associated 
only with osteomalacia among diseases of de- 
mineralization, may be a significant influence or 
factor in some cases of osteoporosis. 

BoTie Metcibolism, 

Kadioisoptic measurements of calcium kmetics 
in association with metabolic balance studies have 
been in use in tlxis laboratory over the past five 
years as indicators of calcimn metabolism and of 
the mechanism of influence thereon of various 
physiological factors. In addition to measure- 
ments of readily miscible pool size, a mathematical 
term, Cae, has been calculated, representing the 
rate of disappearance of tracer from the circulat- 
ing pool after correction for urmary and fecal 
excretions. This value is assumed to represent 
the rate of incorporation of isotope mto bone by 
the processes of new bone formation, of exchange 
with crystal surface calcium, and of exchange with 
more imreactive crystal calcium. Undoubtedly, 
this number may be slightly in excess of the "true" 
value for rate of calcium uicorporation into bone, 
since no corrections are being made at present for 



losses in sweat, but it does provide a function for 
comparison. 

A. Mechanism of effect of elevated dietary cal- 
cium intake in osteoporosis. The results of 
twenty-one radiocalcium studies in five patients 
with osteoporosis have thus far been analyzed. As 
has already been reported by this laboratory, Cas 
values in osteoporosis mider control conditions 
were not significantly different from those in nor- 
mal subjects. This finding has suggested that if 
the rate of incorporation of calcimn into bone in 
this disease is normal, the process by which de- 
mineralization comes about over several years time 
may weE be increased resorption, a suggestion re- 
cently supported in part by microradiographic 
studies. With respect to the influence of increased 
dietary calcimn intake in osteoporosis, analysis of 
combined isotope and balance data have suggested 
the following conclusions : 1) increased dietary in- 
take of calcium over several weeks led to increased 
absorption and retention of calcium; 2) since the 
miscible calcium pool did not increase, the addi- 
tional calcium absorbed was deposited in the body 
outside of tliis readily available pool, most prob- 
ably into bone, in "^aew of the extent and duration 
of the calcimn retention; 3) the gradual decrease 
in rate of incorporation of calcium into bone, Cas, 
with successive isotopic studies on increased cal- 
cium intake may be explained by the filling or 
saturation of osteones (bone units at the micro- 
scopic level) with mineral, others having shown 
that in "untreated" osteoporosis osteones are rela- 
tively unsaturated with mineral; and 4) since in- 
creased retention of calcium on increased calcimn 
intake was not associated with an elevated Cas, 
the retention can probably be best accomited for on 
the basis of decreased bone resorption. 

B. Mechanism of effects of various factors on 
bone metabolism in osteitis deformans (Paget's 
disease of bone) — preliminary results. Osteitis 
deformans is characterized clinically by very rapid 
bone destruction and reformation, the rate and 
extent of which is roughly indexed by a higlily 
elevated serum alkaline phosphatase. Calcium^5 
kinetics studies in six patients readily confirm this 
rapid turnover, indicating a large pool of readily 
exchangeable calcium, a high Cae and retention 
of a large fraction of administered isotope. Alter- 
ations in these indices upon administration of 



218 



ANNTIAI, REVIEW OF INTRAMURAL RESEARCH 



various factors suggest that corticosteroids de- 
crease the excessive rate of bone formation and 
that androgenic-anabolic steroids have the reverse 
effect. Increasing the dietary calcium intake re- 
sulted in no change in serum alkaline phosphatase, 
an increase in the exchangeable pool and a decrease 
in CaB and in the fraction of isotope retained; 
it is not clear as yet whether these latter findings 
indicate that increased calcium intake decreases 
bone formation or merely tends to saturate with 
mineral steones which ordinarily form so rapidly 
that they take up mineral poorly. 

C. Electrolyte and mineral metabolic effects of 
synthetic adrenal cortical steroids. To date thir- 
teen metabolic balance studies of the effects of five 
adrenocorticosteroids have been carried out in 
eleven individuals, including normal subjects, 
patients with rheumatoid arthritis and patients 
with generalized osteoporosis accompanying rheu- 
matoid arthritis. These studies have each neces- 
sarily required several weeks, including an average 
of 33 days on the steroid plus lengthy pre- and 
post-drug control phases, because of the sluggish 
shifts of calcium as compared with the response of 
other elements to hormonal action. In summary, 
although the majority of the patients developed 
increased urinary calcium on the corticoids, the 
effects of fecal calcium were quite varied. In the 
normal subjects and in the patients with rheuma- 
toid arthritis alone, fecal calcium usually in- 
creased, resulting in negative calcium balance. On 
the other hand, in patients with rheumatoid 
arthritis and osteoporosis, fecal calcium was de- 
creased resulting in no change in calcium balance 
or in more positive balance. The locus of this 
difference in effect, whether at the bone or intes- 
tinal mucosa, is as yet imdetermined. In addition 
to the above reported studies, during the past year 
a third 90 day metabolic study has been completed 
of the effects of a new synthetic, 6-alpha-fluori- 
nated corticosteroid, but analyses are not yet 
complete. 

D. Citrate metabolism studies in various bone 
diseases. Kinetic studies of citrate metabolism 
were undertaken because of the known high citrate 
content of bone and the likelihood that citrate may 
be importantly involved in the processes of bone 
resorption. Data obtained utilizing a constant in- 
fusion technique in 15 patients with bone disease 
and 8 normal subjects were analyzed with respect 



to a two compartment model. Computation was 
made of four major variables : apparent volume of 
distribution, primary mixing pool size, net frac- 
tional rate loss from this pool, and turnover of 
the pool. The following significant deviations 
from normal in citrate kinetics were noted: in 
osteoporosis, an increase in fractional rate loss; 
in severe Paget's disease, a three-fold increase in 
pool size and turnover; upon administration of 
adrenal corticosteroid, an increase in fractional 
loss rate ; in hypoparathyroidism, a decreased turn- 
over ; and in idiopathic hypercalciuria, a decreased 
pool size and turnover. Certain of these findings, 
particularly those in Paget's disease, coincide with 
what would be anticipated from previous knowl- 
edge of bone metabolism in these disorders, but 
assessment of significance must await more de- 
tailed analysis. 

Energy Metabolism 

Studies of energy metabolism involving use of 
the Metabolic Chamber for continuous analysis of 
respiratory gas exchange over extended periods of 
time continue to be involved in characterization 
and analysis of obesity from both the physiological 
and metabolic point of view. Certain factors have 
become evident which contiiibute to a small but 
significant extent to the maintenance of obesity 
once established, notably pronomiced phj^sical in- 
activity under most circumstances and less increase 
in energy expenditure during exposure to cold than 
normal individuals exhibit. Energy balance ob- 
servations, involving precise measurement of 
caloric intake and energy expenditure, have not as 
yet substantiated the idea of a "liypophagic 
obesity" type, as described by others, but patterns 
of energy expenditure analyses suggest the pos- 
sibility of significant metabolic differences among 
obese individuals and between certain obese 
individuals and normal subjects. Studies also 
continue of important temperature regulation 
processes, including the interrelationships between 
metabolic response to cold, body insulation and 
body fat content, and the mechanism of response 
to cold exposure in patients with various types of 
fever. 



Obesity 

A. Energy balance during weight reduction 
During the course of weight reduction studies in 



4 



mi 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



219 



obese patients it has become clear that total energj 
expenditure is reduced dramatically as weight is 
lost. Eeductions of energy expenditure as large 
as 1200 Kcal per day have been observed. Each 
kilogram of body weight lost in the various sub- 
jects has been associated with a reduction in en- 
ergy expenditure of from 30 to 10 Kcal per day. 
This pronounced decrease in energy output with 
weight loss is probably associated with two fac- 
tors: 1) reduction in physical work required to 
move the body mass, and 2) loss of metabolically 
active tissue. The contribution of both factors 
is currently being assessed. 

Not only was total energy expenditure over 
the whole day reduced upon weight reduction con- 
sequent to caloric restriction, but resting metab- 
olism both awake and asleep was also reduced. 
In most patients studied the reduction in resting 
metabolism occurred, as one would expect, in pro- 
portion to the decrease in surface area or in other 
units of reference of body tissue in common use. 
In certain obese individuals, however, the reduc- 
tion in resting metabolism was greater than would 
be expected. Since a similar observation had been 
noted previously with semi-starvation at similar 
caloric levels (600-1000 Kcal/day) in individuals 
of noiinal body composition who had minimal or 
no fat stores available for energy (U. of Minne- 
sota), it is suggested that some obese individuals 
respond metabolically to diet restriction as though 
semi-starved even though obviously literally sur- 
rounded by abundant energy-loaded fat. Im- 
paired fat mobilization and utilization may thus 
be one of the metabolic problems in certain types 
of obese individuals. 

B. Metabolic characteristics of obese individ- 
uals. A limited number of biochemical analyses 
made during the course of weight reduction 
studies have shown a fall in total serum choles- 
terol, and increases in serum and urinary ketones, 
the ketone changes being accentuated during 
phases in which exercise was added to the regi- 
men. Inter-individual variation was noted in 
other responses, some subjects showing a fall in 
serum phospholipids and rise in fatty acids. Ee- 
sponses to the administration of glucose also 
varied considerably. 

C. Contribution of physical activity to weight 
reduction during caloric restriction. The con- 
tribution of physical exercise to negative caloric 



balance has been assessed at several levels of 
caloric intake. Interpretation of these results has 
been complicated by the fact that caloric com- 
pensation for imposed physical activity (daily 
walks) occurred during the remaiuing or non- 
walking hours of the day. This compensatory re- 
duction in caloric expenditure was observed even 
when activities for the remaining hours were con- 
trolled. Compensation was never quite complete, 
however, and regular exercise still contributed 
significantly to negative caloric balance. The 
data suggested that the less the dietary caloric re- 
striction, the greater the relative contribution of 
exercise to negative caloric balance and hence to 
weight loss. 

The obese patients observed to date (both by 
visual observation and by scrutiny of continuous 
records of oxygen and carbon dioxide exchange) 
appear to lead distinctly hypokinetic lives. This 
hypokinesis apparently may be induced by the 
obese state and subside with the latter's removal, 
as suggested by one successfully reduced male and 
one female who appeared to increase their spon- 
taneous physical activity in proportion to the ex- 
tent of their weight loss. On the other hand, 
most patients thus far observed have not changed 
their physical activity habits as a result of weight 
loss. Two groups are suggested: 1) Inactive — a 
state perpetuated by obesity and lessened by 
weight reduction ; enhanced mobility through ex- 
tensive weight loss permits these individuals to 
perform activities they were formerly unable to 
do, for example, such simple things as bending 
over and tying shoelaces; and 2) Hypokinetic — 
lack of normal stimulus to move or be moved, or 
a lack of "appetite" for physical activity of any 
kind. 

The relatively small daily energy expenditure 
associated with the hypokinetic state which tends 
to maintain overweight is fostered by the follow- 
ing impressive behavior ; inactivity compensatory 
to imposed physical activity, use of body support 
whenever possible (e.g., when walking on the 
treadmill, a firm grip on the attached upright 
support was found to be necessary) and routine 
assmnption of energetically economic body posi- 
tions. In addition to these physical means, the 
saving of calories by the obese is enhanced by a 
metabolic device not frequently called into play : 



643301—62- 



-16 



220 

a metabolic response to cold whicli is minimal or 
absent. 

Oold and Body Fat Content 

It was demonstrated previously that an inverse 
relationship existed between body insulation and 
the metabolic response of near nude subjects to 
cold air. Insulation, however, was not completely 
identifiable with either total body fat or subcuta- 
neous body fat. Subsequent investigation has re- 
vealed that obese subjects who lose considerable 
amounts of obesity tissue on caloric restriction 
follow the formerly established prediction equa- 
tion for increasing metabolic response to cold as 
they lost weight and insulation. Therefore, no 
distinctive metabolic irregularities in obese sub- 
jects have been identified which suggest abnormal 
hypothalamic regulation of body temperature or 
heat production with implications for associated 
changes in hunger or satiety mechanisms. 

Periodic Fever and Energy Metabolism in the 
Cold 

Investigations of the metabolic and vasomotor 
response to cold air exposure have been carried out 
in patients with periodic fevers of natural (per- 
iodic Mediterranean fever) and experimental 
(malaria) origin, since both groups symptomati- 
cally suggest poor cold tolerance, the question 
arose as to whether temperature regulation against 
cold would be modified either transiently or per- 
manently in these disease states. If modification 
of regulation should be seen, what inferences could 
be made concerning the mechanism of the aberra- 
tion? The following is tentatively suggested by 
the limited data in hand : a) experimental malaria 
decreases the metabolic response to cold but recov- 
ery of a more normal response occurs within 3 
weeks following the febrile period; b) one bizarre 
case of periodic Mediterranean fever failed to re- 
spond metabolically to cold while his body cool- 
ing patterns were remarkably a typical and highly 
variable, indicating either a lesion in or a lack of 
sensitivity of the central temperature regulating 
mechanism; and c) two periodic fever patients 
have responded to cold within normal limits when 
afebrile. There has been the suggestion in one 
patient that exposure to cold stimulated the oc- 
currence of a febrile attack when the exposure to 



ANNUAL REVIEW OF INTKAMTJRAL RESEARCH 



cold occurred in the "sensitive" period before an 
expected febrile period. 



i 



HEMATOLOGY UNIT 






Blood Diseases 

Neonatal throTribocytopenic purpura 

Using the sensitive techniques developed for 
measuring immunoreactions involved in post- 
transfusion purpura (see publication), the syn- 
drome of neonatal thrombocytopenic purpura was 
studied in six children born of four normal 
mothers. For the first time, it was clearly demon- 
strated that maternal immunization against anti- 
gens on fetal platelets was responsible for throm- 
bocytopenia in the infant. Neonatal thrombocy- 
topenic purpura was found to be in eveiy way 
analogous to erythroblastosis fetalis, in that 
mothers who themselves lacked an antigen which 
was present on fetal platelets by inheritance from 
the father, developed either blocking or comple- 
ment-fixing antibodies against the platelet anti- 
gen. The antibody, transmitted through the pla- 
centa, was capable of destroying platelets in the 
fetus and newborn. The appropriate form of 
therapy to be used in cases of isoimmune neonatal 
purpura is not clear as yet, for effects of steroids, 
splenectomy and other forms of treatment on the 
natural course of the disease camiot be evaluated 
without regard to the nature and level of the 
responsible antibody. With the recognition of 
different specific antibodies and means of measur- 
ing different types of antibody activity, evaluation 
of therapy will be simplified and is at present 
under study. 

Platelet-antigen systems 

Over the years there have been numerous at- 
tempts to establish platelet groups in the same 
manner that red cell groups have been established, 
but none of the platelet antigen systems reported 
so far have withstood the test of time. The iso- 
antibodies found in this laboratory to be the cause 
of post-transfusion purpura and neonatal purpura, 
as well as isoantibodies which have developed in 
individuals receiving multiple transfusions, have 
permitted these NIAINID investigators to identify 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



221 



and characterize clearly the nature and inheritance 
of at least seven platelet antigens. All of the 
antigens identified by these antibodies are in- 
herited as codominant alleles ; all of the antigens 
have differed from known erythrocyte antigens, 
and so far there has been no indication of link- 
age between these antigens and erythrocyte anti- 
gens or other inherited characteristics. By using 
quantitative complement fixation procedures and 
quantitative adsorption techniques, this laboratory 
has shown that the amount of antigen per platelet 
is directly proportional to gene dose. Thus they 
have been able to translate directly genetic pheno- 
types into genotypes and identify three genotypes 
on the basis of reactions with each antibody. The 
ability to genotype individuals directly has sim- 
plified considerably the determination of gene fre- 
quency as well as the differentiation of the various 
platelet groups. As a result of fiinding so many 
different platelet-antigen groups, these workers 
have proposed a systematic nomenclature (see 
publication) which we hope will prevent the con- 
fusion which has arisen in erythrocyte-antigen 
notations. 

Platelet transfusions 

During the past seven years there has been much 
interest in the possibility of replacing platelets in 
individuals who have thrombocytopenic purpura, 
due either to bone marrow failure, as in aplastic 
anemia, or to suppression of megakaryocyte activ- 
ity by malignant cells, as in leukemia. Eecent 
studies elsewhere have suggested that on a statis- 
tical basis, thrombocytopenic patients transfused 
with large amounts of platelet concentrates tend 
to bleed less frequently than untreated patients. 
In none of these studies, however, has an attempt 
been made to assess the function of transfused 
platelets by such tests for platelet function as clot 
retraction activity and correction of the bleeding 
time. In a series of patients, including leukemics 
and individuals with aplastic anemia, this labora- 
tory has determined the survival of transfused 
platelets as well as the effects of transfused plate- 
lets on the bleeding time and clot retraction activ- 
ity. In four of these patients platelets survived 
at what appeared to be therapeutically effective 
levels by platelet count, and the clot retraction of 
the patients' blood was corrected, but their bleed- 
ing time was not corrected. Since the bleeding 



time is perhaps the best test for the physiologic 
effectiveness of platelets, it appears that more is 
to be gained by evaluating the physiologic effec- 
tiveness of transfused platelets, than simply by a 
statistical analysis of bleeding tendency in large 
groups of patients who may or may not bleed re- 
gardless of therapy. These studies also indicate 
that packing of platelets, wliich is necessary when 
numerous normal donors are used to provide plate- 
let concentrates, markedly decreases the effective- 
ness of transfusion primarily by diminishing the 
yield of circulating platelets. The haphazard re- 
sponse to packed platelet transfusions reported by 
other investigators appears to be due simply to 
the unpredictable effects of concentrating plate- 
lets, rather than to antibody formation or to 
changes in responsiveness of the patient. 

TJie mosaic pattern of sites for attachment of 
antibodies on cell surfaces 

A proposed model for antibody attachment and 
complement fixation on cell surfaces, described by 
equations derived by T. Hill (collaborator at Uni- 
versity of Oregon, see previous publication) was 
found to be applicable to the problem of deter- 
mining the precise geometric pattern of antibodies 
attached to cell membranes. From data obtained 
on the amount of complement fixed when relative 
concentrations of antibodies and cells were varied, 
and from calculations made using various proxim- 
ity factors in Hill's equations, it has been possible 
to show that drug antibodies attach to cell surface 
sites which are arranged like the four corners of 
a quadrangle, that cells which contain one gene- 
dose of antigen for isoantibodies have antigenic 
sites arranged in a linear fashion, and that cells 
with two gene-doses of antigen have antigenic sites 
arranged as two parallel lines in close proximity. 
The ability to determine the spacial arrangement 
of antigenic sites on cell surfaces may have funda- 
mental implications concerning the structure of 
cell membranes and the manner in which different 
types of antibodies cause cellular injury. 

Anti-hemophilic factors 

The thromboplastin generation test, which is the 
best technique so far devised for measuring anti- 
hemophilic factors (AHF), does not permit meas- 
urement of these factors when they are present in 
vivo in concentrations of less than 5% of normal. 



222 



ANlSrUAL REVIEW OF INTEAMURAL RESEARCH 



Therefore, measurement of the in vivo half-life 
of anti-hemophilic factors has been possible only 
after relatively large amounts of these factors have 
been infused in vivo. The half-life of AHF ap- 
pears to be in the order of 6 to 10 hours and of 
plasma thrombophastin component (PTC) in the 
order of 12 hours by this method. From numer- 
ous measurements of changes in the clotting time 
and prothrombin consumption produced by infus- 
ing minute amounts of anti-hemophilic factors in 
five patients with AHF deficiency and two pa- 
tients with PTC deficiency, it was foimd that 
amounts of these factors in the range of .02 to 1% 
of normal produced correction of clotting time 
and prothrombin consumption which was propor- 
tional to the concentration of the factors. Stand- 
ard curves relating the degree of correction of 
clotting time and prothrombin consumption to 
the in vivo concentrations of anti-hemophilic fac- 
tors in the range of 0.02 to 1% permitted a simple 
assay for determining in vivo survival of these 
factors after infusing amounts equivalent to 0.25 
to 1% normal concentration in hemophiliacs. The 
intravascular "half-life" of AHF by this method 
was found to be 8 hours, the "half -life" of PTC, 
24 hours. After infusing large amounts of anti- 
hemophilic factors, the initial decay could be 
measured by the thromboplastin genertion test, 
and the subsequent decay from 1% to 0.02% by 
correction of prothrombin consumption. It was 
found that the initial decay of AHF and PTC 
was more rapid the larger the amount infused, 
but that subsequent decay of very low levels of the 
factor was greatly prolonged. Thus, it appears 
that once the extravascular compartments are sat- 
urated with anti-hemophilic factors, the decay of 
AHF is in the order of 12 hours instead of 6 to 8 
hours, and the decay of PTC is in the order of 3 to 
4 days, instead of 1 day. This information con- 
cerning the nature of extravascular stores of anti- 
hemophilic factors and the tnxe biological "half- 
life" of the factors pro^ades a basis for developing 
better therapeutic regimens with anti-hemophilic 
agents currently available, and the new method 
of assay will permit evaluation of therapeutic 
agents which may be developed. 

Gamma- gloTyulin 

Studies of the nature of the reaction of a gam- 
ma-globulin which arises in otherwise normal indi- 



viduals and appears to react stoichiometrically : 
with AHF to inactivate this factor completely (see 
1960 report) have been directed at measuring the , 
survival of the gamma-globulin In vh-o and the 
molar concentrations of AHF-anti-AHF involved 
in in vivo and in vitro reactions. The minimum . 
concentration of anti-AHF required to inactivate 
all circulating AHF in a normal individual has 
been determined by in vivo infusions of the . 
inhibitor, and the survival of the inhibitor in vivo 
has been determined. These studies not only pro- 
vide information concerning the molar concentra- 
tions of a clotting factor which has been too labile 
to purify, and the production rate of the factor, 
but also indicate the intensity and degree of 
plasma relacement therapy necessary to treat the 
disease caused by the inhibitor. 

Refractory anemias 

Lack of production of erythropoietine does not 
appear to be an etiologic factor in "refractory" 
anemias. High concentrations of eiythropoietine 
have been demonstrated in 23 of 25 patients with 
"refractory" anemia ; in two patients the results of 
the bioassay were borderline. There also appeai-s 
to be a correlation between the hemoglobin level, 
functional capacity of the stem cell and erythro- 
poietine concentration. In general, a patient with , 
a f mictionally intact stem cell compartment has a 
lower erythropoietine level at a given hemoglobin 
concentration than does a patient with refractoiy 
anemia, where the stem cell compartment is af- 
fected. Presumably this difference reflects utili- 
zation of erythropoietine. 

Only one of nine patients treated with testos- 
terone has responded with increased red cell pro- 
duction ; this patient had an acquired hypoplastic 
anemia and the white cells and i^latelets were un- 
affected. The difference between the results with 
testosterone in this laboratoiy and those reported 
in the literature may reflect a difference in the eti 
ology of anemias which morphologically and 
chemically may appear similar. 



PEDIATRIC METABOLISM BRANCH 

During the year 1961 problems selected for in- 
vestigation in the Pediatric Metaljolism Branch' 
were cystic fibrosis of the paiicreas, other diseases 



NATIONAL rNSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



223 



leading to malabsorption in children and disorders 
due to glycogen storage. 

Cystic fibrosis of the pancreas has constituted 
the objectives of many of the studies and much of 
the effort conducted in this Branch. During the 
past year a total of 110 patients with fibrocystic 
disease have been studied at the NIAMD, many of 
them referred from faraway States or even from 
abroad for further iavestigation of the diagnosis 
and treatment of tliis condition. Of this number 
62 were hospitalized on the Pediatric Metabolism 
service and the remainder followed in the out- 
patient department of the Clinical Center. 

Cystic Fibrosis of the Pancreas 

Cystic fibrosis of the pancreas is a generalized 
disorder in which there is a widespread dysfunc- 
tion of exocrine glands. Abnormalities m exo- 
crine secretions include such varied findings as an 
abnormal physicochemical behavior and chemical 
structure of mucus and electrolyte abnormality in 
the composition of eccriiie sweat and saliva. The 
common denominator responsible for the wide- 
spread dysfunction of so many and perhaps all 
exocrine glands and therefore the pathogenesis of 
cystic fibrosis has thus far escaped detection. It 
is generally agreed that the basic defect in this dis- 
order whatever its nature is genetically trans- 
mitted probably as a recessive trait. 

The investigations on this disease in the PSCB 
have been conducted with the ultimate aim of elu- 
cidating the basic defect and thus establishing a 
better and more logical basis and approach to 
treatment of tliis disease. 



Genetics and Electrolyte Abnormality 

It is generally agreed that the basic defect in 
cystic fibrosis is transmitted as an inherited charac- 
ter. There is no general agreement, however, 
about the mechanism of transmission thought by 
most investigators to be a recessive trait but en- 
visioned by others as an autosomal dominant 
character. 

Studies have been pursued on evidence which has 
been gathered in the previous year m this Branch 
on the effects of varying genetic endowment on the 
diverse manifestations of the disorder itself. It 



has been thought that eccrine sweat defect in pa- 
tients homozygotic for the gene of cystic fibrosis 
consists of two separate features : one, a high level 
of sweat electroyltes and, two, lack of the normal 
decrease in sweat clilorides and sodium concentra- 
tions in response to stress (e.g. : dietary salt re- 
striction, administration of steroids, environmen- 
tal heat) . Evidence has been imcovered by us that 
a proportion of the heterozygotes for the gene of 
cystic fibrosis may show abnormally high levels of 
sweat electrolytes, but are capable of decreasing 
the sweat electrolyte concentration in response to 
stress situations. 

It is becoming clear, therefore, that the un- 
known metabolic or enzymatic defect which may 
be the common denominator of the generalized 
exocrine dysfunction in cystic fibrosis may be 
absent in varying degrees in homozygotes, heter- 
ozygotes, and possibly intermediary cases accord- 
ing to the genetic endowment. Tliis is true of 
other inherited errors of metabolism (e.g.: sickle 
cell anemia, galactosemia and others) . If a simi- 
lar mechanism obtains in cystic fibrosis it may ac- 
comit for some of the many variations in the de- 
gree of involvement and in the severity of 
the clinical picture. 

The "sweat test" is used throughout the world 
in the diagnosis of cystic fibrosis of the pancreas. 
In an effort to determine further the values in- 
dicative of this disease, sweat levels determined 
in more than 1,000 control children and 550 pa- 
tients with cystic fibrosis were analyzed for vari- 
ous parameters, such as effect of various types 
of stimulation and many others. A better and 
more precise definition of the values considered 
to be pathologic and the ones which are within 
normal limits were thus obtained greatly con- 
tributing to the diagnostic usefulness of these 
determinations and enliancing the value of this 
test as a tool for genetic and clinical investigation. 
(Gibson, di Sant'Agnese, Jones and Powell) 

The possible relation of cystic fibrosis in the 
pediatric age group to many of the common gas- 
trointestinal and pulmonary disorders of adults 
has been pursued. This is one of the challenging 
problems at the present time and is actively under 
study in this Branch in collaboration and in cor- 
respondence with many of the principal investi- 
gators in this country and abroad. 



224 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Mucus and Mucopolysaccharide 

During the year of 1961 studies have been con- 
tinued to elucidate and if possible to uncover the 
basic defect in mucopolysaccharide metabolism 
presumably responsible for many of the manifes- 
tations of the generalized disease. Investigations 
have been pursued by analytical methods in vari- 
ous body secretions. Leads uncovered previously 
as to the occurrence of distinctive mucopolysac- 
charide fractions have been pursued and further 
definition of their abnormal composition has been 
sought. In addition, studies have been continued 
to try to clarify the factors involved in the un- 
usual physicochemical behavior of mucopolysac- 
charides, previously described. Investigations in 
the biosynthesis of mucopolysaccharides by study- 
ing the nucleotides involved have also been 
actively continued. 

Previously it was shown by our group that in 
the duodenal contents of fibrocystic patients there 
is a mucopolysaccharide fraction which is easily 
denatured and rendered insoluble in water. 
Wliereas this substance is present in almost all 
samples from patients, it does not obtain in any 
of the samples from controls. In collaboration 
with Dr. Dische, Department of Biochemistry, 
Columbia University, further investigations along 
these lines have been continued and work is pro- 
ceeding on isolation and further analyses of these 
mucopolysaccharide components by a variety of 
different methods including continuous-flow elec- 
trophoresis, chromatography, fractionation with 
organic solvents. Factors which may explain this 
abnormal behavior are being further defined. 

In collaboration with Dr. G. Ashwell, Labora- 
tory of Biochemistry and Metabolism, NIAMD, 
studies on the biosynthesis of mucopolysaccharides 
have been actively continued. A number of com- 
pounds have been isolated and the structure and 
composition of some sugar nucleotides not isolated 
heretofore have been described. 

A program has been initiated to determine the 
distribution of phosphorylated metabolites in t